枣疯病抗性的分子机制研究
摘要
枣(Ziziphus jujuba Mill.)是我国特有的宝贵种质资源和重要的特色果树树种。枣疯病(Jujube witches’broom disease,JWB)是由植原体(Phytoplasma)引起的高致死性传染病害。抗枣疯病品种的选育已经取得了一些进展,但抗病机制研究尚处于起步阶段。本试验以前期课题组选出的抗枣疯病骏枣品系‘交2’为试验材料,对其嫁接到冬枣和赞皇大枣砧木上出现的抗病性状差异和嫁接后在相同砧木上同种接穗间出现的抗病性状差异进行基因组和转录水平分析,以期在分子水平探讨抗病性产生差异的可能机制,为鉴定抗病基因及后续的抗病育种奠定基础。获得的主要结果如下:
1.明确了DNA提取过程中苯酚抽提对枣疯病病原检测有显著影响。利用苯酚抽提与否获得的基因组DNA进行枣疯病病原检测和AFLP分析,发现苯酚抽提后,在感染枣疯病病原的基因组DNA中,病原检出率降低,尤其是病原含量较低时,甚至检测不到病原;但苯酚抽提与否对AFLP分析的扩增效果及图谱没有明显影响。
2.确定了赞皇大枣、冬枣等12个品种寄主中枣疯病植原体的分子分类地位。通过对16S rDNA保守序列的分析,证明赞皇大枣、冬枣寄主中枣疯病植原体都可归于榆树黄化组(16SrⅤ组),二者同源性达到99.86%。
3.初步明确了‘交2’在表观症状上枣疯病抗性出现差异的分子机制。AFLP分析表明,‘交2’在不同品种砧木上出现的表型差异应该是由于砧木基因型对接穗产生了影响导致的;cDNA-AFLP分析表明,‘交2’在同种砧木上出现的抗、感表型差异是因为在RNA表达水平产生了显著差异。
4.通过AFLP分析,获得了5条枣疯病抗性相关序列,这些片段都与毛果杨cc-nbs-lrr抗性蛋白的mRNA同源性达到了70%以上。这5条DNA片段中,4条为嫁接到冬枣砧木上‘交2’接穗中的特异条带,1条是嫁接到赞皇大枣砧木上‘交2’接穗中的特异条带。
5.通过相同砧木上出现不同抗病表现的‘交2’的cDNA-AFLP分析,得到了94条差异条带,其中8条与嫁接后期特异性表达相关;74条与抗病机制的充分表达密切相关;12条生长发育持续表达相关条带。其中,7条序列与大肠杆菌BL21(DE3)中葡萄糖基转移酶、DNA指导的RNA聚合酶等序列同源性均达到97%以上;1条序列与葡萄contig vv78x075502.6同源性为78%;2条序列与葡萄contig vv78x205215.10全序列同源性均为78%;2条与毛果杨预测蛋白mRNA同源性都为72%。
Chinese jujube (Ziziphus jujuba Mill.) is an important and native fruit tree of China. Jujube witches’broom (JWB), caused by phytoplasma, is one of the most serious and destructive disease of Chinese jujube. Selecting cultivars with high resistance was regarded as the most powerful method to eradicate JWB and had made some progress,but the mechanism of resistance was still in its beginning stage.
‘Jiao 2’, a strain of Ziziphus jujuba Mill.‘Junzao’, showed different resistance to JWB when it was grafting on diseased‘Dongzao’and‘Zanhuangdazao’. In this paper,‘Jiao 2’was used as material to study the molecular mechanism of the resistance to JWB. The different mechanism was analyzed on the molecular level and the result would provide foundation for cloning of resistance gene and resistance breeding. The main results were as follow:
1. Using phenol during the DNA extraction had significant affect on the detecting of JWB phytoplasma. Comparing the result of detecting of JWB phytoplasma and AFLP between using phenol and unusing phenol,the detection rate of JWB phytoplasma was decreased and even JWB phytoplasma couldn’t be detected when the amount of JWB phytoplasma was in lower level. But using phenol during the DNA extraction had no significant effect on the result of AFLP analysis compared with the contrast.
2. The molecular taxonomic status of JWB phytoplasma of‘Dongzao’and‘Zanhuangdazao’was defined. Based on the analysis of 16SrDNA conserved sequence,it proved that the similarity of JWB phytoplasma from‘Dongzao’and‘Zanhuangdazao’was 99.86% and both of them belonged to 16SrⅤgroup.
3. The different resistance mechanism of‘Jiao 2’to JWB was identified preliminarily. According to the result of AFLP analysis, the scions with phenotypic discrepancy when they were grafted on different diseased rootstocks were caused by the genotypic difference of rootstocks. The result of cDNA-AFLP showed that the scions with phenotypic discrepancy in the same rootstock were caused by the differential expression on RNA level.
4. Based on the result of AFLP, 5 bands of cc-nbs-lrr gene which related to the resistance were gained. Among them,4 bands were from‘Jiao 2’grafting on diseased‘Dongzao’and 1 band was from‘Jiao 2’grafting on‘Zanhuangdazao’. The similarity between the sequences of those bands and mRNA of cc-nbs-lrr was more than 70%.
5. 94 bands of differential expression were gained by cDNA-AFLP analysis of‘Jiao 2’with phenotypic discrepancy on the same rootstock. 8 bands expressed specifically in later period of grafting; 74 bands were related to the expression of the resistance mechanism; 12 bands were related to growth and development of jujube. The result of blast showed that 15 sequences had high similarity(more than 97%)with E.coli BL21(DE3);3 sequences had similarity of 78% with Vitis vinifera contig VV78x205215.10;2 sequences had similarity of 72% with mRNA of Populus trichocarpa.
1.明确了DNA提取过程中苯酚抽提对枣疯病病原检测有显著影响。利用苯酚抽提与否获得的基因组DNA进行枣疯病病原检测和AFLP分析,发现苯酚抽提后,在感染枣疯病病原的基因组DNA中,病原检出率降低,尤其是病原含量较低时,甚至检测不到病原;但苯酚抽提与否对AFLP分析的扩增效果及图谱没有明显影响。
2.确定了赞皇大枣、冬枣等12个品种寄主中枣疯病植原体的分子分类地位。通过对16S rDNA保守序列的分析,证明赞皇大枣、冬枣寄主中枣疯病植原体都可归于榆树黄化组(16SrⅤ组),二者同源性达到99.86%。
3.初步明确了‘交2’在表观症状上枣疯病抗性出现差异的分子机制。AFLP分析表明,‘交2’在不同品种砧木上出现的表型差异应该是由于砧木基因型对接穗产生了影响导致的;cDNA-AFLP分析表明,‘交2’在同种砧木上出现的抗、感表型差异是因为在RNA表达水平产生了显著差异。
4.通过AFLP分析,获得了5条枣疯病抗性相关序列,这些片段都与毛果杨cc-nbs-lrr抗性蛋白的mRNA同源性达到了70%以上。这5条DNA片段中,4条为嫁接到冬枣砧木上‘交2’接穗中的特异条带,1条是嫁接到赞皇大枣砧木上‘交2’接穗中的特异条带。
5.通过相同砧木上出现不同抗病表现的‘交2’的cDNA-AFLP分析,得到了94条差异条带,其中8条与嫁接后期特异性表达相关;74条与抗病机制的充分表达密切相关;12条生长发育持续表达相关条带。其中,7条序列与大肠杆菌BL21(DE3)中葡萄糖基转移酶、DNA指导的RNA聚合酶等序列同源性均达到97%以上;1条序列与葡萄contig vv78x075502.6同源性为78%;2条序列与葡萄contig vv78x205215.10全序列同源性均为78%;2条与毛果杨预测蛋白mRNA同源性都为72%。
Chinese jujube (Ziziphus jujuba Mill.) is an important and native fruit tree of China. Jujube witches’broom (JWB), caused by phytoplasma, is one of the most serious and destructive disease of Chinese jujube. Selecting cultivars with high resistance was regarded as the most powerful method to eradicate JWB and had made some progress,but the mechanism of resistance was still in its beginning stage.
‘Jiao 2’, a strain of Ziziphus jujuba Mill.‘Junzao’, showed different resistance to JWB when it was grafting on diseased‘Dongzao’and‘Zanhuangdazao’. In this paper,‘Jiao 2’was used as material to study the molecular mechanism of the resistance to JWB. The different mechanism was analyzed on the molecular level and the result would provide foundation for cloning of resistance gene and resistance breeding. The main results were as follow:
1. Using phenol during the DNA extraction had significant affect on the detecting of JWB phytoplasma. Comparing the result of detecting of JWB phytoplasma and AFLP between using phenol and unusing phenol,the detection rate of JWB phytoplasma was decreased and even JWB phytoplasma couldn’t be detected when the amount of JWB phytoplasma was in lower level. But using phenol during the DNA extraction had no significant effect on the result of AFLP analysis compared with the contrast.
2. The molecular taxonomic status of JWB phytoplasma of‘Dongzao’and‘Zanhuangdazao’was defined. Based on the analysis of 16SrDNA conserved sequence,it proved that the similarity of JWB phytoplasma from‘Dongzao’and‘Zanhuangdazao’was 99.86% and both of them belonged to 16SrⅤgroup.
3. The different resistance mechanism of‘Jiao 2’to JWB was identified preliminarily. According to the result of AFLP analysis, the scions with phenotypic discrepancy when they were grafted on different diseased rootstocks were caused by the genotypic difference of rootstocks. The result of cDNA-AFLP showed that the scions with phenotypic discrepancy in the same rootstock were caused by the differential expression on RNA level.
4. Based on the result of AFLP, 5 bands of cc-nbs-lrr gene which related to the resistance were gained. Among them,4 bands were from‘Jiao 2’grafting on diseased‘Dongzao’and 1 band was from‘Jiao 2’grafting on‘Zanhuangdazao’. The similarity between the sequences of those bands and mRNA of cc-nbs-lrr was more than 70%.
5. 94 bands of differential expression were gained by cDNA-AFLP analysis of‘Jiao 2’with phenotypic discrepancy on the same rootstock. 8 bands expressed specifically in later period of grafting; 74 bands were related to the expression of the resistance mechanism; 12 bands were related to growth and development of jujube. The result of blast showed that 15 sequences had high similarity(more than 97%)with E.coli BL21(DE3);3 sequences had similarity of 78% with Vitis vinifera contig VV78x205215.10;2 sequences had similarity of 72% with mRNA of Populus trichocarpa.
引文
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