Transfer, Identification And Expression Of Barley α-Amylase Inhibitor Gene Isa-H1 In Wheat

The wheat pre-harvest sprouting is a worldwide natural disaster. Periods of rain during pre-harvest may cause unharvested wheat to sprout. In this course the activity of many hydrolytic enzymes is elevated, especially high level cx-amylase. It is aamylase that hydrolyze the endosperm starch reserves that severely limit the use of such grain for manufacture. A bi-functional ct-axnylase and subtilisin inhibitor from barley has been shown to inhibit wheat a-amylase, and the gene (Isa-Hi) codes for the inhibitor of endogenous of a-amylase and subtilisin inhibitor is located on the long arm of barley chromosome 2. Our goal is to transfer this gene into wheat and to study the performance. The results are as follows:1.The genetic constitution of fifteen SC2 materials derived from tissue culturegeneration of the cross CSphibxCS+Betzes 2H were analysed, and two nullisomic/ 2H trisomic lines X99-1 and X99-3, two monosomic alien substitution lines X99-2and X99-9, six disomic alien substitution lines X99-4, X99-5, X99-7, X99-11, X99-12 and X99-14, and one substitution / translocation line X99-13 were screened by(3ISH.2.The wheat-barley disomic alien substitution lines 2H(A), 2H(B) and 2H(D) were identified by CS 2A, 2B, 2D double ditelocentric lines (DDT) and RFLP analysis. X99-4 and X99-5 were 2H(A), X99- 11, X99- 12 and X99- 14 were 2H(B) and X99-7 were 2H(D). The wheat chromosome 2B was substituted by barley chromosome 2H in the substitution / translocation lines. The RFLP probe psrl3l on the short ann of wheat homoeologous group 2 can be used as molecular marker to tag barley chromosome 2.3.The frequencies of cells with 2n=2 iii in pollen mother cells at meiotic I (PMCs MI) for 2H(A), 2H(B) and 2H(D) were 92.05%, 94.12% and 95.49%.Which indicated that the substitution lines 2H(A), 2H(B) and 2H(D) were stable incytology.4.The barley chromosome 2H has good genetic compensation for the wheat chromosome 2B and 2D with respect to spike length, spikes per plant and other agronomic characters. There were some different in fertility, and the level of seed set for 2H(A), 2H(B) and 2H(D) was 59.66%, 79.70% and 78.18%.5.The wheat-barley 2H monosomic line was obtained from SC1 of the cross CSphlb x CS+Betzes 2H with high frequency 12.5%. The SC1 and SC2 of the cross 2H(B)xCS were screened by STS primer aABC252 which located on the long arm and aABC454 for short arm of barley chromosome 2, and the frequency rate of transmitting barley chromosome 2 was 90% and 50% in SC1 and SC2. The two recombination materials 19-1-1 and 19-1-5 were selected from 74 SC2 plants and the chromosomal configuration of recombination material 19-1-1 in PMCs MI is 2n~20II+I+t+t.6.The barley a-amylase inhibitor was purified by aninionium sulfate precipitation, ion exchange on Sepharose, and isoelectric focusing(IEF). The result of inhibiting experiment indicated that the complex was formed between BASI and wheat a-amylase- 1, and the activity of wheat a-amylase- 1 was reduced, and other results showed that BASI could also inhibit the activity of wheat a-amylase-2.7.The ASI content was studied by IEF, and the results indicated that each line began to synthesis ASI 15 days after flowering and reach the highest content at 30 and 35 days. The ASI concentration of addition lines CS+Betzes 2H and the substitution lines 2H(A), 2H(B) and 2H(D) were higher than that of CS, and it showned that the gene Isa-HI can express well in wheat background.8.There are some problems of gene interaction, expression and coordination after transferring barley chromosome 2 into wheat. The euploid substitution lines 2H(A), 2H(B) and 2H(D) were better than addition line CS+Betzes 2H at plant vigour and starch quality. The substitution line 2H(A) had good effect in defendingwheat pre-harvest sprouting, and the 2H(B) had good starch quality owing to it抯 high peak viscosity.