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Functional Characterization of the Gene

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Welcome to the course of Plant Developmental Biology.So, in previous class, we we were discussing the reverse genetics based approaches, andwe have already finished how to analyse or how to monitor gene expression pattern.And then now in this class, we are going to discuss about how to functionally characterisea gene.So, to functionally characterise a gene which means that you want to know what is a specificfunction of a gene in a particular developmental process.So, how will you know that?So, one approach is always to know some ones function is loss-of-function.So, if you somehow disrupt the gene, and then see what kind of developmental defect yousee when you make a loss-of-function of a gene.And this will tell that for what function your gene is necessary.And then another complimentary approach to validate it or to understand the functionof a gene is the gain-of-function.So, normally let us assume a developmental gene is expressed in a particular tissue,but it is not expressed in some other tissue.Then if you take this gene and ectopically express in the tissue where it was not presentnormally, and then you can look that tissue can it reinitiate some developmental program.And if it is; if this is true, then we can say that a particular gene is sufficient fora particular function.So, these are the two major approach which we take to understand function of a particulargene in development.So, we will take one by one.So, first we will take loss-of-function.And here there are two way to do it either you knock-out the gene or you knock-down thegene.In knock-out what you basically do you totally make a null mutant where the gene is totallydisrupted.In knock-down you are reducing the expression of a particular gene, but it is not null mutant.And in gain-of-function or there are two way to do that, one which I told is ectopic overexpression or ectopic expression.So, you express this gene, you change the promoter, you use some broader promoter toexpress this gene in a tissue where normally it is not expressed, and second method isactivation tagging which we will see in later on.So, first we will take the loss-of-function how to generate knock-outs.So, if you look the first two approaches, this we have already and also discussed inthe in the forward genetic based approaches which is to generate insertional inactivationlines for a particular gene.So, one was the T-DNA insertion, second was the transposon mediated insertion.But the difference between forward genetics and reverse genetics is that in forward geneticswe were not fixing the gene, we were fixing the function or if we were fixing the phenotypeor if we were fixing the biological process, and then we are trying to look for T-DNA insertionlines or transposon insertion lines and then try to map that which gene it is.But for in the reverse genetics, what we do that now we know a specific gene now we knowthat this is the gene and I want to understand what is its biological function, then youcan specifically go for and look for that gene is there some T-DNA insertion lines availablefor that gene or transposon lines available for that gene or not.Another and now very widely used method is genome editing.So, here you are basically editing at the genome level the gene, and then you are generatingknock-outs or loss-of-function mutants.So, first we will take the insertional inactivation lines.So, as I said that the initially people have done at a very global level, they have generateda large number of T-DNA insertion lines or transposon insertion lines, and those lineshas been deposited to some databases.So, for example, there are already so many databases or so many laboratory or so manyprojects they have generated a large number of T-DNA insertion lines or transposon insertionlines.And this all mutants has been preserved and protected.And what you can do that, you can choose any of the mutant, you can request, you can order,and you will directly get those mutants for your detailed functional analysis.So, there are many many large number of this kind of collection of mutants are available.I will take one of the examples.So, for example, this T-DNA express or this is a Salk Institute they have generated largenumber of T-DNA insertion line.What simply you have to do this website is available, you just chose your gene, put thegene ID, search with the gene, search with the chromosome, search with the some of thename or anything, and then they will show a list of the insertion lines available forthat particular gene, and you can chose.Sometime what happens that you might have more than one insertion lines in your gene.And then you can look you can define where you want to have the insertion, for example,if you look this gene you can have so many insertion you can choose that I want one insertionline in exon 1, I want one insertion line in exon 2.And then you just take these numbers and request from the database, and these seeds the mutantArabidopsis seeds will be available to you, and then you can get this mutant or knock-outlines for your detailed functional characterisation.And the first thing what you do, if you get this kind of insertion line, you first establishthat actually there is insertion first thing.Second thing is that what is the genetic pattern is it insertion is homozygous, both the allelesare disrupted or it is heterozygous only one alleles are disrupted.How you can do?This is very easy since you know the site of insertion predicted site of insertion,you know the sequence of T-DNA, you know your gene id you know essentially the sequenceof your gene, you can design a forward primer here, one reverse primer here and one primeryou can design in the T-DNA and then you can do some PCR.If you do with this genomic DNA forward and reverse primer, if there is insertion, ifthere is insertion, the insertion line will not give it because the fragment size is verybig, you will not get the PCR amplification.So, if you have a homozygous line, in homozygous line is basically your both the genomic locushas the insertion.Then if you use LP and RP together you will not get a band here.But if it is wild type or heterozygous, in heterozygous one locus has the insertion,another locus does not have insertion.Then from the locus where you do not have insertion you will get this band.So, basically your wild type and heterozygous both will give band with the LP and RP.Then you choose your LP with this or RP with this depending on in what orientation yourT-DNA is, and then if you have a heterozygous you will have a kind of band here.And then if it is wild type, you will not have this T-DNA specific band.So, this is very important for you to establish that what is the insertion status of thisT-DNA in your gene.Then another important thing what we try to do is that we always back cross because sinceall these has been generated through a random insertion, there is a possibility that inyour in your plant there are more T-DNA insertion site.So, you might have a insertion in your gene, but there might be some other insertions somewhereelse in other genes.And that you can remove by taking these lines crossing with the wild type and then goingto the F2 generation and again screen for homozygous.And then once you have established this line, you can take these lines for detailed functionalcharacterisation or study the developmental defect in this T-DNA or loss-of-functionalanalysis.Another method which is genome editing.So, in genome editing, there are many techniques available which you can use to edit the genomeZFN, then TALEN, but recently CRISPR Cas 9 has been used very widely and very extensivelythese days.So, we will see how this is being used to edit the gene.So, essentially what is this CRISPR Cas 9 based genome editing tool.So, here what we are using?We are usingbacterial endonucleases which is RNA guided endonucleases that is Cas 9.So, and then you what you do simply you produce a guide RNA and you produce Cas 9 proteinin the plant.This guide RNA you can target to your sequence wherever you want.So, you can design a very targeted guide RNA.And then here when you express guide RNA along with the Cas 9 protein, so may be that willbe clear here.So, what you do let us assume this is your gene and you want to edit this gene, you wantto disrupt this gene, what you can do you can, you can choose a target sequence typicallyhere.And there is one requirement which is called PAM sequence.So, you can target your region which is near to the PAM sequence, and you can generateguide RNA which is specific for this sequence.And then you clone this guide RNA in a vector and the same vector you can clone Cas 9 andthe endonuclease protein.And then when you take this and make a transgenic plant both the both the partners will expressthere.Then what will happen this is a guide RNA, this is Cas 9 protein, when Cas 9 proteinwill bind with the guide RNA, guide RNA will target this endonucleases in the in the sequencespecific manner.And when this endonucleases is recruited on the genomic copy, it will make a double strandedbreak in the genomic copy, and then that this break has been considered as a damaged DNA.And endogenous mechanism which is called non homologous end joining mechanism will be activatedin the plant and it will fill this gap, it will repair here.But during the process of this it is end joining mechanism there is a possibility that youcan generate a deletion, you can generate a insertion, you can disrupt the coding region,and that is how you will have a genomic disruption of a particular gene.So, this is some of the method which is being used for making the knock-out lines.Another mechanism what we are using to study the loss-of-function is knock-down or downregulation.So, here we are not disrupting the genomic copy, but we are regulating after the transcription.So, we are reducing the expression level either at the protein level or at the transcriptionlevel.And there are some of the techniques which is being used for this function; one is theanti-sense RNA technology, this was one of the the pioneer technique which was been used,but these days there are many more techniques.So, the second method is double stranded RNA interference or artificial micro RNA.So, in anti-sense RNA what happens that if you have a gene and you know the gene sequence,you just take the same gene or the gene specific fragment of the gene and clone in the anti-senseorientation.Then what are you expecting.So, if it is a genomic copy of your gene, it will transcribe in this direction, butnow you have generated another transcript which is complimentary to this endogenoustranscript because it is anti-sense oriented and then this transcript will go and it willbind with the endogenous transcript, and then this transcript either it will be degradedor it will be inhibited for protein synthesis and you will have the phenotype.This is one example that there are few genes has been targeted through anti-sense RNA,you can see, this is the promoter and the genes has been and the gene specific regionhas been cloned in the anti-sense orientation.And when you have drive this genes, you can basically suppress the endogenous gene usingthis method as you can see the phenotype.So, this is control, when the genes has been suppressed, there are lot of developmentaldefects.And when you check the RNA level or the transcript level, you can clearly see that as comparedto controls in this anti-sense lines, there is significant reduction of the transcriptlevel.But there are some something important if you look this line, here almostdown regulated,but these lines are partial down regulation.So, this is not a kind of null mutant, but this is a kind of down regulation.So, you can have here a gradient or you can have some lines where you have near to thenull phenotype, but some lines you might have which are weak lines, there the down regulationis not so efficient.Second method which is being used for down regulation is double stranded RNA interferencetechnology.This technology were developed from the endogenous mechanisms.So, in plants siRNA and micro RNAs are known as a wellregulatory mechanism.There is a large number of small RNAs, and they are regulating expression of many manyimportant endogenous gene.So, if you look a general mechanism, there are two way either you have a double strandedRNA or hairpin RNA, this will undergo the process of processing through DICER or AGOmediated processing, then uploading.This I will not go in the detail; I am sure you would have already studied.And then it generate a very small 21 to 24 nucleotide single standard antisense RNAs.And these RNA they can go and pair with endogenous messenger RNA, and then they will either targetthat messenger RNA for the degradation or they can inhibit protein synthesis or theycan do epigenetic regulation any kind of regulation is possible.So, to do that, if you want to use this technology, so what you can do you can generate your ownRNA interference construct for a specified genes.What you have to do?You have to simply mimic this hairpin generation, how you can do, you can do you can do takea gene specific fragment and clone in sense orientation, and then put some non-specificsequence here or stuffer fragments here, linker fragment, and then you take same the samefragment and clone downstream, but in opposite orientation.If this is in the sense orientation, this you put in the antisense orientation and puta promoter here, drive this hair pin loop.Then what will happen?This messenger RNA which will make it will make a this kind of a structure.So, this region and this region will pair here.And it will generate a hairpin structure, and this will be endogenously processed andthen it it will generate siRNA.And this siRNA specifically will target the endogenous RNA or messenger RNA against whichit has been designed, and then it will down regulate the expression of those genes.There are plenty of examples were RNA interference has been used to down regulate the gene.Some of them I will show here.So, if you look here two of the genes has been down regulated by RNA interference basedapproach.You can look here this is northern blot in control there is high level of expression,but in RNA interference line the level has significantly decreased.And you can see the phenotype panicle phenotype.Similarly, this is a very important transcription factor MADS 1.And when the RNA interference line was generated for MADS1 you can see this is northern blotagain.In wild type, you have a very high level of expression, but in RNA interference line theendogenous expression is almost null.Whereas you can detect the hair pin structure which is basically bigger of the size.Not only that if you go and do a very spatial northern blot, you can detect small siRNAsas well.So, these are the small siRNAs which is not present in the wild type, but which is presentin the RNA interference line.So, this has been very extensively used to down regulate many many of the genes.Then third method which is been used to make a knock-down mutants are artificial microRNA.So, as I said micro RNA is a natural small RNA present in the genome of the plants, theyare being processed through dicer and ago mutated mechanism.And then this micro RNA, they are going and targeting specifically some of the genes,and regulating its expression at different level, transcriptionally, post transcriptionally,translationally, epigenetically.And then this technique was taken and then used to generate some artificial micro RNA.So, basically what you do that if you take a natural hairpin structure or the loop ofthe naturally existing micro RNA, and if you can replace this core sequences the microRNA core sequences which is basically showing the complementarity with the gene.And if you replace with your own gene of interest, can you down regulate it.And then this can be easily done by combination of multiple PCR.So, for example, if this is your construct here you can use some different set of primersyou can amplify this region from here to here, then you can amplify this region from hereto here, and you can amplify this region from here to here.So, basically this red is your artificial micro RNA sequence.Then if you use these three as a template together and use this two primers, so youcan combine them in this way.And then this artificial micro RNA, you can clone under promoter and then generate someknockout line.And if you see here this has been quite successfully used in some of the study.For example, if you the take this example of Arabidopsis, in Arabidopsis, this is leafymutants, so this is basically insertional mutant.So, if you look LEAFY12, this has a significant phenotype as compared to the wild type.But if you target this LEAFY through artificial micro RNA against the LEAFY, you can mimicthe phenotype.So, your artificial micro RNA here is as strong as the other mutants.Similar is the case if you look ft mutant.So, ft mutant, if you look the wild type, the plant is already flowered it has achievedthe reproductive stage whereas, this ft mutant has very delayed flowering.But if you generate again artificial micro RNA against the FT, you can see that it isshowing a severe phenotype as the other mutant.So, this tells that artificial micro RNA is one of the very efficient way to down regulategenes in Arabidopsis.And similar kind of things has been used in other plant species.For example, if you take the case of rice.So, here artificial micro RNA has been generated for couple of genes.So, this is SPL11, and this is PDS gene.And you can clearly see this is the control and this is the RNA or artificial micro RNAline and you can see clear phenotype in artificial micro RNA lines.So, basically; so through this study either you go through the knock-out based study oryou go through the knock-down based study.What you conclude that you can generate a loss-of-functional mutants and you concludethat this genes are necessary for this particular function.Another complimentary way as I said that is gain-of-function.So, first way that we can generate ectopic over expression or ectopic expression construct.Here what you do, you use some broader promoter or a promoter which is expressed at the highlevel.And you can use either tissue specific promoter for the tissue where your gene endogenouslydoes not express or you can use a kind of general 35S promoter or Ubiquitin promoterto drive your gene in the sense orientation.And then you can have high-level of expression in the tissue where you do not expect yourgene endogenously present.And if you look the cases in this mutant, you can clearly see that this is your basallevel of expression, but when you have over expressed under 35S promoter, you can seethat in most of the lines the expression level is very high as compared to the control.And you can see here the relative expression and then this is co related with the phenotype.So, this is and then if you can go to the next generation, you can clearly see thisis one of the line has been tested in the next generation, so may be here the linesis segregating.If you look this plant, it is not showing phenotype, but these two plants are showingthe phenotype.And if you go and check the over expression level, it clearly co relate.So, this plant which is not having phenotype does not show the over expression as well.But these two plants which show the over expression, they show the phenotype.Similar kind of analysis has been done for the transcription factor MADS1.And you can clearly see when MADS1 was ectopically over expressed in transgenic rice, you cansee a very significant phenotype in the panicle architecture and at the spikelet development.And if you see the expression level, you can clearly see that in the control you do nothave expression in this tissue, but these lines they have a very very high level ofexpression.So, another way to generate a gain-of-function mutant is activation tagging.In activation tagging basically what you do you take T-DNA construct, and you just putsome multiple copy of enhancer elements.Here you can take 4 x copy of the 35S enhancer element.And then randomly throw in the genome and it will go and integrate, but what happensthat if it comes in a in a proximity with some of the basal promoter or some of thepromoter which normally is expressed at the very low level or normally it is not expressed.But now the promoter has got some of the enhancer element in the close proximity and then thepromoter will start activity it will start very strong expression.And in that case, what will happen that you will see a phenotype which could be becauseof the gain of function phenotype.These are some of the example.So, if you look here, there is a insertion of enhancer elements here.And if you look the result of this is that if you look this gene, this gene is more expressedin the lines insertion lines than the control.So, if you look here, and then you can see this there are some of the phenotype of thisline.So, basically this is called enhancer tagging, you can look for many more genes and identifymore activation tagging lines and you can take them for the studying the function .So, this is a typical way to functionally study gene in plant, but there is some problem.So, let us assume that if you want to study function of some of the essential genes, whathappens, if you make a knock-out or if you make a knock-down, if gene is essential youface a problem of lethality.If it is lethal, if loss-of-function is lethal or gain-of-function is lethal, you will nothave transgenic plant or you will not have a plant to study.In that case, how to solve the issue?Some of the genes are very important at very early stage during embryo development.So, they could be embryonic lethal some of the gene might be not embryonic lethal, butthey could be lethal at the seedling stage or at any stage they complete their vegetativegrowth, but they cannot enter in the reproductive phase.So, basically they will not make a fertile flowers, they will not make seeds which meansthat you cannot go to the next generation.So, if you want to study such kind of genes through the reverse genetics based approachhow to do it.In that case, what you have to do you have to use a regulated gene expression based approaches.So, you cannot take a gene or you can down regulate all the time or you can down regulateconstitutively or you can over express constitutively.If you do that, you will not be able to study.Then what you can do, you can generate a construct or you can make a strategy where you can specificallyregulate a gene whenever you want or you can have a kind of more temporal and spatial basedregulation of your gene silencing or overexpression.So, there are three way how you can regulate gene typically one is the spatial regulatedconstruct.So, what you can do that instead of over expressing a gene in all the tissues, you target overexpression in a some of the tissue where you feel that might not be so harmful that itwill it will have lethality.For such kind of things you can use some tissue specific promoters or known promoter whichis expressed in a particular organ, for example, you can check in the leaf and you can usesome leaf specific promoter, and express a flowers in the leaf and look can it startsome of the flower specific developmental program in the leaf.Second way of regulation what you can do is the temporal regulation construct.Here what you can do you want to express or down regulate a gene at a particular stagenot before that.For example, let us assume that down regulation of a gene X is embryonic lethal, which meansthat if you down regulate from day one even embryogenesis will not complete, then youwill not have plant.In that case, what you can do, and if you want to study the role of this gene maybein the vegetative development or maybe in the reproductive development, then you donot need to down regulate or silence or over express this gene during the process of embryogenesis.So, what you can do, in that case you can you some stage specific promoter to drivethe construct over expression construct RNAi construct or any construct.And then what you can do you can skip your early stage of embryogenesis.So, embryogenesis will occur normally then later on you just down regulate and see whatis the consequence.Second is that you can even combine both and you can generate a construct where you cando both together.So, you can regulate a gene or a construct over expression, RNAi construct temporallyas well as spatially.How you can do it?We will take just one example of both the cases.For example, if you are studying a transcription factor, there is a system which is being verywidely used is a glucocorticoid receptor - based system.So, we know that transcription factors, they has to enter inside the nucleus for activatingthe gene expression.And if you somehow retain them in the nucleus and if you can regulate their translocationbetween cytoplasm and nucleus, you can regulate their activity and that is being done by fusingyour transcription factor with a rat or mouse glucocorticoid receptor.So, glucocorticoid basically is a steroid hormone in animals and what happenes thatthe glucocorticoid receptors they are normally present in the cytoplasm, when they receivesignal through the hormones, then what happened that they only then enter inside the nucleusand go and activate their genes.So, if I take a gene here is the example of MADS 1; if I take MADS1 gene and fuse withdelta GR and overexpress under any broad promoter does not matter here, 35S promoter.Then what will happen under normal condition, I have a high level of MADS1 expressed, proteinis already made fusion protein, MADS1 delta GR, fusion protein is already made it is ready,but it is only present in the cytoplasm.Since, MADS1 is a transcription factor until and unless it will enter inside the nucleus,it cannot perform the function.Then if I take this plant and induce with hormone which we are using here dexamethasone,then what will happen it will enter in the nucleus and it can activate the gene.And this is very important technique, which you can use to even distinguish between directgenes which are regulated directly by the MADS1 or the gene which are consequence ofthe MADS1 induction.So, if you combine this dexamethasone treatment with the protein synthesis inhibitors cycloheximide.So, let us assume if this is example of OsMGH3; OsMGH3 as I said earlier that this is oneof the gene which is regulated by MADS1.And what happens, when you treat with the dexamethasone you can see at a particulartime point the expression level of OsMGH3 is going up, which means that MADS1 is sufficientto activate expression of OsMGH3.But when you treat with the cycloheximide, so basically cycloheximide is a protein synthesisinhibitor.So, it will not allow new protein to be synthesized, in that case whatever MADS1 protein is alreadymade delta GR protein is already made, only that will enter inside the nucleus and itwill activate immediate genes, but those genes which are getting activated at the transcriptionlevel those RNA will not make protein, because there is cycloheximide protein.And if protein is not made, there target will not be activated.So, the MADS1 indirect target will not be activated, but direct target will be activatedand that is how you can see here, when we treat dexamethasone and cycloheximide togetheryou cannot see the activation of OsMGH3.This tells that OsMGH3 is not directly regulated by MADS1, but it is indirectly regulated byMADS.And then if you look this construct, this is another very important construct.So, this is a case when you down regulate MADS1 gene what happens that it is all thefloral organs are defective, you will not make a fertile seeds.You can see here this is the floral organs, they all are getting converted into leaf likestructure or kind of lemma palea like structure and you will not have organ.So, it is very challenging to the next generation is almost impossible.In that case what you can do?You can make a MADS1 delta GR fusion as well as artificial micro RNA.And then what you do, normally this artificial micro RNA will suppress the gene you havethe phenotype, but if you grow in presence of dexamethasone this delta GR will go andit will complement the phenotype.So, this construct has over expression as well as artificial micro RNA, but importantthing here is that this artificial micro RNA is designed to target 3 prime untranslatedregion of the MADS1 and that 3 prime untranslated region is not present here.Here we have taken MADS1, sorry here we have taken MADS1 only till the stop codon, untranslatedregion is not present why, if we take that region, then this artificial micro RNA willgo and silence this gene as well.So, in this construct this micro RNA will specifically silence the endogenous copy ofthe gene, but it will not silence the transgene copy and this way you can use a complementationbased assay and do what whatever, phenotypic analysis or you can go even identify the downstreamtarget, direct downstream target to study.You can see here, when you induce with dexamethasone protein is getting localised to the nucleus,but when you do not induce, you do not see the protein inside the nucleus.Another very important way, technique which is being used to regulate both temporal aswell as spatial expression, simultaneously is XVE bases system or estrogen receptor – basedsystem.So, here basically what has been done.So, one chimeric transcription factor had been generated, so this XVE; XVE is a chimerictranscription factor, here there are three component one is X, V and E.X is a DNA - binding domain of LexA protein, LexA is from the bacteria.And then V is trans-activation domain of VP16 protein which is from virus.And then E is the human estrogen receptor.