The Allelic Variation And Mechanism Of Starch Synthase Ⅱa(SSⅡα) In Chinese Landrace Wheat

Starch is major component as 50%-70%of bread wheat seeds thus its composition directly determine wheat yield and its composition and physicochemical properties which can affect flour processing quality.Starch synthase Ⅱa(SSⅡa)is an important enzyme in the biosynthesis of starch in amylopectin synthase and the activity of SSIIa directly influence the content of amylopectin and starch.In this study,we evaluated the diversity of SSⅡa proteins in 368 Chinese landrace wheat by using SDS-PAGE.Gene cloning and expression level measurement were conducted to analysis the effects on starch content of the allelic variation of SSⅡa.The major results were described as follows:1.SSII-D null type was identified in three accessions(Xiaoqingmang、Pushanbamai and P119)out of all 368 Chinese wheat landrace by comparison of types of Triticum monococcum,A egil ops tauschii,Triticum t.urgidum and nullisomic-tetrasomic lines of Chinese Spring.However,none of SSⅡa-B null line was detected in tested lines.2,A total 8262bp SSlla-D genomic sequence was isolated,and included 1367bp upstream of start codon,6756bp from ATG to TGA and 138bp downstream stop codon.Alignment of SSⅡa-D genomic sequence showed that the upstream sequence of in Pushanbamai was completely consistent with that in Chinese Spring,whereas the 18bp deletion and 9bp variation was detected in the junction region of 7th intron and 8th exon,the 18bp deletion was existed in the end of 7th intron and the 9bp variation was existed in the start of 8th exon region when compared with control line.This variation has led to the deletion of intron-exon junction recognition site AG and four types cDNA was detected though amplification of cDNA of SSlla-D gene sequences.For type Ⅰ and Ⅱ,the splicing sites were moved to 8th exon region that leading to the variations of cDNA length at corresponding region(type Ⅰ:65bp deletion;type Ⅱ:16bp deletion);for both type Ⅲ and type Ⅳ,the splicing sites were advance to 7th intron region that leading to different degree of cDNA length longer:a 39bp less deletion at 7th intron,9bp variation at 8th exon for type III was observed,while 183bp less deletion at 7th intron,9bp variation at 8th exon was detected for type Ⅳ.Furthermore,another 97bp that less cut in the end part of 5th exon was also detected in the type IV cDNA sequence.The results of amino acid translation revealed that the premature termination was found in type I,Ⅱ and IV type cDNA.In addition,13 amino acid residues was inserted in type III amino acid when compared to that in control lines.In addition,a total of 9 type II cDNA sequences were obtained in all 12 sequences,while only one for each another three types were acquired,suggested that the major transcriptional pattern of SSlla gene in Pushanbamai was type II.3.Sequence analyses of SSlla-D cDNA in Xiaoqingmang indicated that AC was inserted at 4th exon region,which led to frameshift mutation and thus incomplete amino acid sequences was translated.However,two types of SSIIa-D were amplified for P119 cDNA sample,of which typel with six positive clones was completely consistent with that in Xiaoqingmang,in addition,a 97bp insertion at +1221 bp was detected in typeⅡ with one positive clone,which was consistent with type IV variation in corresponding region in Pushanbamai.4.No significance was detected in total starch content and amylose content among Xiaoqingmang.Pushanbamai,P119 and all control wheat lines(Chuannongl6,Shumai969 and Chinese Spring),suggested that deletion of SSⅡa-D showed minor effect on amylose content or were implemented by other functional genes related to amylopectin synthesis.5.The results of expression level during seed development stage indicated that the expression level for three SSIIa-D deletion lines were lower than that of control line Chinese Spring at DPA10,DPA17,DPA24 and DPA31.