Functional Analysis Of The Pheophytinase Gene Ta Pph And Mining Superior Allele In Wheat(triticum Aestivum L.)

Leaf senescence is a complex and highly programmed process during in the final stage of leaf growth and development.Premature senescence of wheat leaf leads to damaged photosynthetic system,retarded wheat growth and yield.The most obvious sign of leaf senescence is chlorophyll degradation.Therefore,understanding the molecular mechanism of chlorophyll degradation will improve yield by delaying leaf senescence.In this study,a chlorophyll degradation enzyme gene Ta PPHs was cloned.The function of Ta PPH was identified by transforming rice and analyzing the interaction proteins.This provided a theoretical basis for clarifying the mechanism of chlorophyll degradation in wheat leaf senescence.In addition,wheat is rich in allelic variation,therefore,mining favorable alles in germplasm resources and developing functional markers are cornerstones for germplasm innovation and molecular breeding in wheat.This study also cloned genomic sequence of Ta PPHs,analyzed its sequence polymorphisms,developed functional markers,associated analysis of Ta PPH allele with yield-related traits and chlorophyll content,and explored superior allele.The main results are as follows:1.Chlorophyll degradation key enzyme genes Ta PPH-7A,Ta PPH-7B and Ta PPH-7D were cloned in wheat,the full-length ORF were 1467 bp,1464 bp and 1467 bp,respectively.Ta PPHs contained a conserved α/β-hydrolase domain with serine activity and a PPH motif.Ta PPH located in the chloroplast.2.Ta PPH was highly expressed in wheat leaves,especially in senescence leaves,responding to dark and ABA stress.3.Overexpression Ta PPH-7D accelerated leaf senescence in Arabidopsis.Under ABA stress,chlorophyll degradation was faster in old leaves of overexpression lines than that in wild type(WT),whereas had no significant difference between the overexpression lines and WT in seedling.4.Ta PPH-7D transgenic rice exhibited a more severe leaf senescence phenotype.Chlorophyll degradation was significant faster in overexpression Ta PPH-7D rice than that in WT under natural senescence,dark and ABA stress conditions.Chlorophyll degradation-related genes were significant up-regulated in overexpression Ta PPH-7D rice.5.There was a strong interaction between Ta PPH and Ta CLH,indicating that Ta PPH and Ta CLH may be involved in chlorophyll degradation during wheat leaf senescence.6.The full-length coding region sequence of Ta PPH-7A,Ta PPH-7B and Ta PPH-7D were 4479 bp,4313 bp and 4067 bp,respectively;Ta PPHs contained five exons and four introns,which were located on chromosomes homologous 7 group.Ta PPH-7A contained 4 SNPs and 1 In Del,Ta PPH-7D were highly conserved among wheat diversity panel.7.The SNP at 1299 bp(A/G)was chosen to develop a d CAPS marker Ta PPH-7A-d CAPS.Association analysis showed that Ta PPH-7A-1(A)can positively regulate TGW in different environments and the Chl during grain filling stage under drought stress,and the excellent allele Ta PPH-7A-1(A)was positive selected in Chinese wheat breeding programs.The increased expression of Ta PPHs may be included by the combination of some senescence-related transcription factors and cis-acting elements,which will accelerate the chlorophyll degradation and further accelerate leaf senescence,affect crop yield.In the future,Ta PPH-7A-1(A),a superior allele related to TGW and Chl content,can be used to improve the predictability of parental selection and the accuracy of future generation material selection in wheat hybrid,the efficiency of genetic improvement,and the level of wheat breeding...