Creation And Mechanism Of Transgenic Wheat (Triticum Aestivum L.) Mediated With RHA2b Gene In PHS Resistance

Preharvest sprouting(PHS)in wheat(Triticum aestivum L.)is the germination of grains in the ears when long range rainfall or damp conditions prior to harvest occur.Pre-harvest sprouting is one of the major adverse effects of high yield and stable yield of wheat.Seed viability and hydrolysis of starch and protein in the endosperm are always reduced after PHS.At present,most of the wheat varieties used in the production have certain ear sprouting characteristics.The way to solve the preharvest sprouting was as follows: firstly,the application of chemical control technology;secondly,breeding varieties with resistance to preharvest sprouting.Breeding resistant cultivars is the best way to solve the problem of sprouting,but the selection and application was restricted by limited resistance source.Creating high resistance wheat varieties by molecular transgenic breeding technology is an ideal way to solve the preharvest sprouting.In order to make full use of the RING FINGER genes to improve the resistance to preharvest sprouting in wheat,the RsRHA2b/TaRHA2 b was cloned.The highly efficient and stable way to create transgenic wheat was established.The transgenic homozygous lines were obtained.Based on the transgenic homozygous lines,the effects of the introduction of exogenous RsRHA2 b gene on the key enzyme activity of starch synthesis and protein synthesis,the expression of the related genes were studied.The cDNA library was constructed from the embryo of barley seeds treated with different ABA concentration.The proteins interacted with TaRHA2 b were screened by yeast two hybrid system.At the same time,the new genes of preharvest sprouting resistance were identified.The distribution of TaRHA2 b gene coding region in donor ancestors and different varieties was studied to explore the mechanism mediated with RHA2 b gene in PHS Resistance.The main results are as follows:1 RHA2 b,a radish E3 ubiquitin ligase gene,enhances seed dormancy and tolerance to preharvest sprouting of transgenic wheatThe RsRHA2 b gene was cloned and transferred into Zhengmai 9023 by Agrobacterium-mediated stem apex transformation.The transgenic lines were checked by basta resistance,PCR,RT-PCR,q RT-PCR and Southern blot.Seed germination experiment of transgenic lines was carried out.In the presence of 0.5?mol/L ABA and 5 ?mol/L ABA,the weight of transgnic lines was measured,and the expression profile of genes involved in ABA signaling transduction mediated with RHA2 b,such as ABI5,FUS3 and MAL were analyzed.The results of analysis confirmed that the RsRHA2 b gene was integrated into the genome of wheat and it could be stable.The seed dormancy and PHS tolerance was significantly enhanced in transgenic plants with stable expression of RsRHA2 b.The significant reduction of weight was found between the transgenic wheat plants and non-transgenic Zhengmai9023.Furthermore,the expression of ABI5,FUS3 and MAL genes were significantly accumulated at the second leaf stage of the transgenic wheat plants with ABA treatment.These results displayed that RsRHA2 b gene has the function to improve dormancy and tolerance to PHS in transgenic wheat.2.Conditions for establishment of cell suspension culture of wheat “Zhengmai9023”Rataria of wheat “Zhengmai 9023 ” were cultured to study the effect of different mediums on callus induction and estate of the callus.The results showed that the optimal callus induction medium was MS+ 3.0mg/L 2,4-D,the highest ratio of callus induced was 92.5%;the optimal medium for callus subculture was MS+1.0mg/L 2,4-D + 0.1mg/L KT;the optimal medium for cell suspension was MS+1.0mg/L 2,4-D + 0.1mg/L KT;the optimal time of suspension system for subculture was 14d;the mature embryos in suspension system could develop into normal plants in the basic medium.The results laid a foundation for establishing a rapid and efficient genetic transformation system of wheat.3.Screening TaRHA2b-interactive Proteins by Yeast Two-hybrid SystemThe normalized cDNA library was constructed basing on barley embryos.The bait protein vector p GBKT7-TaRHA2 b was transformed into AH109 yeast cells to detect its self activation.The AH109 yeast cells containing bait and prey were screened by different defect mediums,?-galactosidase chromogenic reaction.The candidate positive clones were performed in vivo and in vitro pull-down experiments to be checked again.The detection results showed that the cDNA library contained 1.1× 106 recombinant clones and that all the inserted cDNA fragments were between1000 bp and 3000 bp.which meets the requirement of the yeast two-hybrid library.We obtained 5000 plasmids and the sequence information of 120 large fragments after large-scale plasmid extraction and sequencing.The bait vector was without self activation function.132 hybrid clones were screened in the SD/-Leu/-Trp/-Ade/-His medium.84 clones were found after ?-galactosidase chromogenic reaction.Finally,the information of 52 sequences was obtained.8 clones were performed in vivo and in vitro pull-down experiments.The results showed that they were positive.The positive proteins interacting with TaRHA2 b are YTH2450,YTH2456 and YTH2476.These results are helpful to explain the specific signal transduction pathways involved in the regulation of TaRHA2 b gene.4.Cloning and Functional analysis of YTH2450,YTH2456 and YTH2476 genesDNA and RNA were derived from barley leaves.The g DNA and cDNA sequence of YTH2450,YTH2456 and YTH2476 genes were amplified.q RT-PCR was used to analyze the tissue specificity and expression pattern under the ABA treatment.Transgenic rice mediated with YTH2450,YTH2456 and YTH2476 genes were carried out.The results showed that YTH2450 and YTH2456 genes had no introns,and YTH2476 gene had 5 introns.Three candidate genes had strong tissue specificity,a significant level response to ABA.The expression vectors of three candidate genes were obtained.Positive transgenic lines were created.The study laid the foundation for further studying the function of candidate genes and the mechanism mediated with RHA2 b gene in PHS resistance.5.Effects of exogenous RsRHA2 b gene on activity of key enzymes and expression of RsRHA2 b related genes in transgenic linesThe activities of key enzymes of starch and protein synthesis and q PCR analysis of genes involved in RHA2 b gene were studied basing on transgenic lines 1477 and non-transgenic “zhengmai9023”.The results showed that the RsRHA2 b gene can increased the activities of SBE?SSS?AGPP?GBSS and GOGAT.But it inhibited the activities of GS.At the same time,the RsRHA2 b gene promoted the expression of YTH311,YTH611,YTH1065,YTH2437,YTH2438,YTH2456,YTH2496,YTH3049 genes,and inhibited the expression of YTH2433 gene.The effects on the key enzyme activities involved in starch metabolism and protein metabolism and the expression of the related genes by RsRHA2 b gene might be the reason for the improvement of transgenic lines sprouting resistance.6.Cloning and Analysis of the RHA2 b Gene in the Ancestor Species and Pre-harvest Sprouting Resistance and Susceptible Cultivars of WheatGenomic PCR cloning strategy was applied to clone RHA2 b sequences from diploid A genome wheat(Triticum urartu),B genome wheat(Aegilops Speltoides),D genome wheat(Aegilops tauschii)and 22 Chinese wheat cultivars.The PHS resistances of 22 Chinese wheat cultivars were identified.The results indicated that the RHA2 b sequences from ancestor species varied from 441 bp to 476 bp,containing143 and 155 amino acids.The difference between donor B group and A/D group is35 bp.They all comprised a “Zinc finger RING-type profile” domain.There is significant difference of PHS resistances among 22 wheat cultivars.But there is a small difference in the RHA2 b sequence of 22 wheat cultivars.However,there are many SNP sites among them.The current information is useful for throwing light on the mechanism of transgenic wheat mediated with RHA2 b gene in PHS resistance.