【作者】 原亚萍；

【导师】 陈孝； 肖世和；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2001， 博士

【摘要】 小麦成熟期穗发芽（Pre-harvest Sprouting，PHS）是世界性小麦自然灾害，指小麦在收获前遇到阴雨或潮湿环境下的穗上发芽。在整个穗发芽的进程中，多种水解酶的活性得到提高，特别是α-淀粉酶活性的提高，对胚乳中淀粉的分解能力增强，严重限制了面粉的加工利用。大麦2H染色体长臂上携带有α-淀粉酶抑制蛋白基因Isa-H1，将这一基因导入小麦并研究其表达抑制情况，结果如下： 1.通过基因组原位杂交，解析了CSph1b×BetzesCS+2H杂种组培SC₂代15份材料的遗传组成，其中X99-1、X99-3为二体缺体2H三体，X99-2、X99-9为单体异代换系，X99-4、X99-5、X99-7、X99-11、X99-12、X99-14为二体异代换系，X99-13为代换易位系。 2.经中国春重双端体DDT2A、DDT2B及DDT2D测交及RFLP分析，鉴定出一套小麦-大麦2H二体异代换系，其中X99-4、X99-5为A代换即2H（A），X99-11、X99-12、X99-14为B代换即2H（B），X99-7为D代换即2H（D）；同时明确了代换易位系中代换所涉及的染色体为2H/2B；小麦第二部分同源群短臂探针psr131可作为追踪大麦2H染色体的RFLP标记。 3.代换系2H（A）、2H（B）和2H（D）在花粉母细胞减数分裂中期Ⅰ（PMCsMI）具21Ⅱ的频率分别为92.56%、94.12%和95.41%，表明3个代换系染色体基本构型为21Ⅱ，在细胞学上是稳定的。 4.代换系2H（B）、2H（D）的穗长、每株穗数、每穗粒数及籽粒大小等农艺性状均好于CS。2H（A）、2H（B）和2H（D）的不育率分别为40.44%、20.30%和21.82%。大麦2H染色体对小麦第二同源群B、D染色体的缺失有较好的补偿能力。 5.CSph1b×CS+Betzes2H杂种组培当代以高达12.5%的频率获得了小麦-大麦2H单体异代换系，对其继续进行选择，在SC₃代获得了2H二体异代换系2H（B）。利用大麦2H染色体上的STS长臂引物aABC252及短臂引物aABC454对2H（B）×CS杂种组培后代SC₁及SC₂进行筛选，2H染色体在各代的传递率分别为90%和50%。从74株SC₂代中筛选出2株小麦染色体与大麦2HL的重组材料19-1-1、19-1-5。19-1-1 PMCs MI染色体构型为 Zn＝20II十I＋t＋。 6．通过硫酸胺沉淀、离子交换及等电聚焦电泳等方法，提纯了大麦a· 淀粉酶抑制蛋白（BASI）。蛋白功能检测结果表明BASI可与小麦a·淀粉 酶－l结合成复合物，从而达到降低小麦a·淀粉酶活性的目的。同时发现BASI 对小麦a－淀粉酶－2也有抑制作用。 7．采用等电聚焦电泳，分析了各材料抑制蛋白（ASI）的动态变化，结 果表明抑制蛋白在花后15天已开始合成，随着发育天数的增加，蛋白含量 逐渐增加，30．35天含量较高。激光扫描结果表明附加系CS＋B6TZeSZH及代 换系的ASI含量高于小麦品种中国春，说明大麦a－淀粉酶抑制蛋白基因Isa－ HI在小麦背景下表达正常。 8．大麦ZH染色体导人小麦背景后存在协调、表达和基因互作问题，从 植株长势和淀粉品质看，整倍体代换系好于附加系。代换系ZH（）穗发芽率 较低，ZH田)的高峰粘度较高，淀粉品质较好。更多还原

【Abstract】 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.更多还原