Establishment Of Multiplex-PCR Targeting Superior Glutenin Subunits And Expression Regulation Of ABA 8'-hydroxylase Gene By RNA Interference

Wheat is one of the major crops in our country. To meet the requirment of improved living standard of people and develop food processing industry, and to improve commercial competition ability in international trade market, quality improvement is a long-term important objective of wheat genetics and breeding program in China.Wheat quality is affected by many factors, among which the composition and number of high-molecular-weight glutenin subunits (HMW-GS) are well known to be closely relative to end product quality of flour. Duing to the lack of the superior HWM-GS, Chinese wheat varieties possess poor processing quality in the past decades. Ax1/Ax2*, Dx5, and overexpressed Bx7 (Bx7OE) are normally associated with superior end-use quality, especially dough strength. So far, Bx7OE allele has not been exploited widely in China wheat breeding program. In combination with the optimization of reaction components and amplification condition, three pairs of PCR primers targeting for Ax1/Ax2*, Bx7OE, and Dx5 genes were used to establish a multiplex PCR for the purpose of enhanced molecular marker-assisted breeding with good reliability and low cost. After validation with wheat cultivars (lines) with known genes, it displayed that the developed multiplex PCR permitted discrimination of these major HMW-GS in a single PCR reaction and agarose gel assay. A total of 89 wheat cultivars and advanced lines from Tibet were tested by the multiplex PCR-based assay. The results showed that the frequency of Ax1/Ax2* and Dx5 each was 12.4%, and only 10.1% of the accessions possess both Ax1/Ax2*and Dx5, whereas Bx7OE was not detected in the accessions. Exploitation of superior HMW-GS will be beneficial to improving wheat quality in this region.Pre-harvest spouting (PHS) is a widespread problem, which result in significant losses in grain yield and decreased end-product quality. Facing to the serious PHS in most wheat production regions of China, to solve this problem is a great task for wheat scientists. Pre-harvest Sprouting is a complex trait affected by both genotype and environment. Seed dormancy is the dominant factor controlling PHS. As a important phytohormone, Abscisic acid (ABA) plays important role during seed development which is closely involved in the onset and maintenance of dormancy, repressing germination. ABA8'-hydroxylase plays a key in the major ABA catabolite. The CYP707A gene encoding ABA8'-hydroxylase has been firstly cloned from Arabidopsis. The CYP707A homologues from wheat,barley and rice share highly similarity. In this study, two endosperm-specific RNAi vectors targeting barley ABA8'-hydroxylase gene(HvABA8'OH-1) were transformed into rice by Agrobacterium-mediated transformation.50 plants were regenerated after selected culture,18 of which were identified as transgenic plants by PCR. Seeds harvested from TO transgenic plants were used for the primary germination test. Results showed that some of seeds displayed germination delay. Further function analysis of transgenic plants is underway, which will help to establish an new gene engineering approach for controlling the cereal crop PHS.