【作者】 范惠玲；

【导师】 孙万仓；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2007， 硕士

【摘要】 自交不亲和性在十字花科植物中广泛存在,其对种质的保纯及产量的进一步提高造成了较大的影响。积极发掘芸苔属及其近缘植物的自交亲和基因资源,克隆控制自交亲和性的相关基因并研究其功能,进而通过其它途径培育、筛选出自交亲和性植物是解决这一难题的最有效手段。本文以五叶期、十叶期、开花前、开花当天和开花后不同生长期的芸芥自交亲和系叶片和柱头为材料,同时以不同生长期自交不亲和系的相应组织为对照,采用30对引物组合进行mRNA差异显示研究,旨在分离、筛选与自交亲和性相关的特异基因。研究结果如下:1、芸芥自交亲和性可能是由一对基因控制的简单隐性遗传性状芸芥自交亲和系与自交不亲和系不同杂交组合后代F1、F2和BC1F1群体的自交亲和性状况研究表明,F1代全部表现自交不亲和,F2、BC1F1代自交亲和性发生分离。对F2、BC1F1群体自交亲和数据的统计和χ2测验分析表明,所有组合调查群体F2代自交亲和与自交不亲和分离比均符合1:3,BC1F1代自交亲和与自交不亲和分离比均符合1:1,表明芸芥自交亲和性是由一对基因控制的简单隐性遗传性状。2、提出了一种有效的芸芥总RNA提取和纯化方法改进异硫氰酸胍法提取的总RNA经DNaseⅠ处理后完全适用于DDRT-PCR分析。其OD260/OD280的比值介于1.9～2.1之间,RNA样品纯度较高,无DNA、蛋白质等污染;非变性高浓度琼脂糖凝胶电泳显示RNA质量较好,为无降解的完整RNA。针对总RNA中痕量DNA的去除,对两种体系进行了比较研究,确定采用不加RNase抑制剂的一种体系: 20μL总RNA; 2.5μL 10×DNaseⅠBuffer; 1μL DNaseⅠ(5U/μL);1.25μL DEPC处理的水;37℃温浴30 min。3、确立了一套适合于芸芥差异显示分析的反应体系及程序对影响mRNA差异显示扩增的因素逐一进行了比较研究,结果表明在25μL mRNA差异显示总体系中使用17.2μL DEPC-ddH2O;1.5μL 10×PCR Buffer (加Mg2+);2.0μL dNTPs(各2.5mM);1.0μL锚定引物H-T11M (10μM);1.0μL随机引物(5μM);1.5μL cDNA;0.8μL Taq DNA聚合酶(2.5U/μL)。DDRT-PCR反应程序为:1)94℃4 min;2)30℃1 min 30s;3)72℃1 min;4)94℃30s;5)42℃1 min 30s;6)72℃1 min;从4)到6)35个循环; 7)72℃10 min,最后所得DDRT-PCR扩增的效果较好。4、芸芥自交亲和基因的组织特异性表达mRNA差异显示扩增结果表明:不论处于哪个生长期的叶片,采用同一对引物组合进行扩增,在SC与SI之间都没有得到差异片段。说明芸芥自交亲和基因的表达不属于组成型表达而是组织特异性表达。5、与芸芥自交亲和性相关的mRNA差异显示片段18对有效引物组合在芸芥自交亲和系与自交不亲和系开花前柱头mRNA差显研究中共扩增出142条带,其中自交亲和系有90条带显现。条带大小主要集中于200～1500 bp之间。自交亲和系较自交不亲和系有差异带存在,能在不同引物对和不同温度下稳定出现的有2条,一条小于100bp,另一条介于750～1000bp之间;自交不亲和系较自交亲和系也有差异带存在,能在不同引物对中稳定出现的仅有1条,分子量为750 bp。芸芥自交亲和系与自交不亲和系开花当天柱头mRNA差显结果发现,自交亲和系较自交不亲和系的差异条带除了在开花前柱头样品中所出现的2条以外,另外还有1条约300 bp的新差异带出现;自交不亲和系也较自交亲和系出现1条大小为500～600bp的差异带。自交亲和系与自交不亲和系开花后柱头mRNA差显结果与开花当天的相似。研究还发现差异条带多出现在以H-T11C为锚定引物的扩增图谱中。总之,在自交亲和系柱头中共分离得到3条稳定的差异片段,这3条差异片段可能与芸芥自交亲和基因有密切关系。更多还原

【Abstract】 Self-incompatibility is a widespread mechanism in flowering plants that prevents self-fertilization and promotes outcrossing. It has great influence on maintenance of germplasm purification and increasing in seed yield.The most effective method to resolve this problem are finding new gene resources of self-compatibility in Brassica and its relatives, analyzing their function and obtaining self-compatible plants, finally.To date, people could not find the nature of self-compatibility from the aspects of self-compatible genes. mRNA differential display technique is an efficient technique, its application in studying on self-compatibility can isolate differential expression genes from specific tissue,so as to understand the molecular mechanism of self-compatibility and apply them in breeding programmes. Leaves and stigmas at 5-leaf stage, 10-leaf stage, before anthesis, on the day of anthesis, after anthesis from self-compatible E.sativa Mill were used, accordingly, the related tissues of self-incompatible lines as control. With 3 anchoring primers and 10 random primers, specific cDNA fragments which may be closely related to self-compatibility were isolated with mRNA differential display technique. The results are as the following:1.Several hybridized combinations were made between self-compatibility lines and self-incompatibility lines, to analyse the hereditary characteristics of self-compatibility in E.sativa Mill. The results showed that all the F1 plants were self-incompatible, and the F2, BC1F1 populations segregated in ratio.Through chi square test, the segregation ratios of self-compatibility to self-incompatibility in observed F2, BC1F1 populations were coincided with the ratio 1:3, 1:1, respectively. It was inferred therefore that the hereditary characteristics of self-compatibility in E.sativa Mill was controlled by one recessive nucleus gene.2.Total RNA extracted by improved guanidine thiocyanate method and treated with DNaseⅠwere found to be applicable to mRNA differential display technique. Its absorbance ratio OD260/OD280 was ranged from 1.9 to 2.1, which demonstrated that RNA samples were free of contamination of DNA, protein. Agarose electrophoresis showed that RNA samples thus-obtained were in good integrity. 3. An economical reaction system of purification of total RNA was put forward in this study. In order to remove chromosome DNA, two different systems were compared. The results indicated that both RNA samples purified could be used as templates for cDNA synthesis. One system without Ribonucleasei inhibitor was chosen, thus its cost was considerably declined.The purification systems was: 20μL total RNA; 2.5μL 10×DNaseⅠB uffer; 1μL DNaseⅠ(5U/μL); 1.25μL DEPC-H2O; 37℃30 min .4. An effective method of mRNA differential display for E.sativa Mill.was established in the investigation.The factors affected the mRNA differential display reaction system was set up.This system was 25μL total volume contained 17.2μL DEPC-ddH2O; 1.5μL 10×PCR Buffer (plus Mg2+); 2.0μL dNTPs (each 2.5mM); 1.0μL anchoring primer (10μM); 1.0μL random primer (5μM); 1.5μL cDNA; 0.8μL Taq DNA Polymerase(2.5U/μL).mRNA differential display steps: 1) 94℃4 min; 2) 30℃1 min 30s; 3)72℃1 min; 4) 94℃30s; 5) 42℃1 min 30s; 6)72℃1 min; from 4) to 6) 35 cycles; 7)72℃10 min.5.The results of mRNA differential display on leaves at 5-leaf stage, 10-leaf stage, before anthesis, on the day of anthesis and after anthesis found that amplified cDNA fragments using every pairs of primers were the same among the self-compatible lines and self-incompatible lines at the same growth period, which showed tissue specific expression patterns of self-compatible genes and that they were not expressed within all the tissues, especially which were not correlated with the state of development of the leaves.6.With 30 pairs of primers, mRNA differential display on stigmas from self-compatible lines and self-incompatible lines before anthesis were performed. 142 cDNA fragments were obtained, of which 90 ones could be seen in self-compatible lines, 52 ones in self-incompatible lines.The great majority of bands amplified were 200～1500bp. Self-compatible lines had 10 differentially expressed bands of which only 2 bands exhibited stably with different pairs of primers,the one was less than 100bp,the other was 750～1000bp; in addition, 6 cDNA fragments appeared in self-incompatible lines, only one 750bp band generated repeatedly.All 133 cDNA fragments were obtained in mRNA differential display products of sigmas on the day of anthesis.Of which 87 ones exhibited in self-compatible lines and 46 ones in self-incompatible lines. Besides two specific bands which existed before anthesis, another 300bp new differential band was found unique to self-compatible lines. At the same time, one 500～600bp band was identified in self-incompatible lines.The results of mRNA differential display on stigmas after anthesis were identical to those observed on stigmas on the day of anthesis. The study also found that anchoring primer H-T11C was more efficient than others in the identification of differentially expressed bands.Sum up, three repetitive and steady differential cDNA fragments were presented in stigmas of self-compatibility,which might be closely related to the self-compatible genes in E.sativa Mill.更多还原