Cloning And Molecular Characterization Of Psy Gene In Wheat Yellow Pigment Biosynthesis And Establishment Of Double Haploid Groups For Yellow Pigment Content

For a long time,color of wheat flour and flour foods have been required highly for people in China.Yellow pigment content(YPC) of wheat(Ttiticum aestivum L. 2n=6x=AABBDD) is one of important factors reducing flour color,and yellow pigment content is significant influence to cooked products and nutritional quality in wheat. Although environmental factors have significant effect on yellow pigment content,gene type is main decisive factors.Yellow pigment content is mixture composed of various substances,and its main components is carotenoids,biosynthesis of carotenoids need Phytoene synthase(Psy),the enzyme is the primary rate-limiting enzyme in biosynthetic pathway of carotenoids,and the gene is first selected gene to carotenoids genetic manipulation.Psy gene has been further studied in many crops(especially maize),but the research has not been developed in wheat.In this paper,wheat Psy gene with different significant YPC was cloned in 248 Chinese wheat cultivars based on maize Psy gene registered in GenBank,and the molecular characteristics of Psy gene and YPC of different wheat cultivars were analyzed too.At the same time,wheat F1 with high and low YPC parents were crossed by maize pollen in order to obtain wheat haploid embryos,then induced wheat double haploid for genetic analysis of yellow pigment content through embryo rescue and haploid chromosome doubling,the purposes are clearing the effect of Psy gene to wheat YPC,developing molecular markers of YPC in wheat,and creating genetic materials and new germplasm for wheat YPC genetic analysis and wheat YPC breeding.The results were as follows:1.Cloning and molecular characterization of Psy gene in wheat yellow pigment biosynthesi Phytoene synthase(Psy) gene,a primary rate-limiting enzyme in the biosynthesis pathway of wheat yellow pigment(YP),was cloned and sequenced through Psy gene of maize and wheat ESTs in this study,at the same time,the Psy gene sequences between wheat and maize were compared.The results showed that,5 DNA fragments with 981bp,751bp,1192bp,690bp and 302bp was amplified by polymerase chain reaction in DNA samples extracted from the high YP and low YP content wheat cultivars,the sequences of high YPC wheat are same as those of low YPC with same primer.Sequences alignment showed that all the 5 DNA fragments were Psy gene(fragment) of wheat,and the full length gene with 6 exons and 5 introns,3437bp was assembled.Detailed molecular characterization of PCR products was analyzed with a case study of primer Psy04,and preliminary locating 5 Psy gene fragments on 7A chromosome in wheat.2.Allelic variation of wheat Psy gene and effect on yellow pigment content Primer Psy02 was designed according to sequence conservative areas of Psy gene in Ttiticum,cloning Psy gene of wheat.The results showed that,3 kinds of PCR products were amplified with 196bp,233bp and 489bp,among them,196bp and 233bp bands cover all sequences of the second exon of wheat Psy gene,37bp difference sequence is a insertion sequence in the second exon of Psy gene,which display different YPC in wheat. Verification test showed,153 samples of 248 wheat cultivars(including 229 micro core germplasms and 19 cultivars with typical quality characters) amplified 196bp band, account for 61.7%,the YPC average value is 7.314mg/kg belonging to high YPC rang,and 95 samples amplified 233bp band,account for 38.3%,the YPC average value is 5.207mg/kg belonging to low YPC rang.Variance analysis showed that the YPC difference reached 1%significant level,which proved the 37bp insertion sequence is one of the reasons of different YPC in cultivars,therefore,primer Psy02 is a important molecular marker identifying YPC,and the gene effects on wheat YPC significantly.Sequence analysis showed that 196bp and 233bp were 100%homologous to primer probe sequence, and the two sequences were located on 7A chromosome.489bp sequence was high homologous to Psy-B1 of EU096094 with 97%identity,and 85%identity to the Psy-A1 of EF600063 on 7A chromosome and DQ642439 on 7B,thus the gene can not lacate on 7A or 7B chromosome.Combining with high YPC of 489bp,conjecturing the Psy genes on 7A and other chromosomes have additive effect to YPC.3.Identifying Psy gene with Aegilops tauschii(Triticum tauschii L.2x=DD=14) and allelic variation Psy gene were cloned by primer Psy01-Psy06 in Aegilops tauschii (Triticum tauschii L.),the results showed that only primer Psy02 and Psy06 amplified specific bands,the PCR product of primer Psy was 206bp,with 93.0%sequence homologous to wheat PCR product,there was only 1SNP in the second exon.And the PCR product of primer Psy06 in Aegilops tauschii was 305bp,with 95.77%sequence homologous to wheat PCR product,there was no SNP in the 6th exon,which showed that there were Psy genes in DD genome of wheat,and the possibilities of locating primer Psy01,Psy03,Psy04,Psy05 and 489bp gene fragment of Psy02 on the D chromosome were eliminated.4.Establishment of DH groups used to study inheritance laws in wheat yellow pigment In order to obtain a large number of wheat haploids to establish the DH groups of wheat yellow pigment content,wheat and maize cross were used to induce wheat haploid embryos and then the haploid plants were produced via young embryo culture.The factors affecting wheat embryos and the regeneration of young plants from young embryos, such as environmental temperature,different period of peeling embryo,size and development stage of young embryos,the treatment time at 4℃and dark treatment time were studied.The results showed:the embryos frequency was the highest when environmental temperature was 21～23℃,there was not significant difference on the frequency of young plants production when haploid embryos was peeled in different time from 12d to 16d;the frequency of young plants production of the size of 0.5～1.0mm is much higher than that of the size of 0～0.5mm and 1.0～1.5mm;the short time treatment between 1～3 days at 4℃would accelerate the regeneration of young embryo,but after 3 days the frequency of callus induction and plants improve the frequency of plants production during the process of young embryo culture.Production would decrease;dark treatment of all 24 hours for 12 days could effectively.And on this condition,a high efficiency and repetitive regeneration system of wheat haploid from wheat×maize was established.