对水稻矮缩病毒传毒能力存在差异的叶蝉群体mRNA差异表达分析
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摘要
水稻矮缩病毒(Rice dwarf virus,RDV),属于呼肠孤病毒科(Reoviridae),植物呼肠孤病毒属(Phytoreovirus),是水稻矮缩病的病原,广泛分布于中国、日本、朝鲜、菲律宾、尼泊尔等水稻种植区,给水稻生产造成严重损失。该病毒可由黑尾叶蝉(Nephotettix cincticeps)作为昆虫介体,它是一种经卵以持久方式传播的病毒。本研究构建了两个黑尾叶蝉群体,它们对水稻矮缩病毒传毒能力有显著差异。并利用mRNA差异显示(differential display RT-PCR, DDRT-PCR)技术对这两个群体进行差异分析,找到差异片段,尝试探讨叶蝉高效率传毒的分子机制。
在构建高低亲和性黑尾叶蝉方面,无毒的黑尾叶蝉经过连续多代饲毒,传毒后,得到了对RDV传毒效率差异明显的两个黑尾叶蝉群体,通过生物学方法鉴定,高亲和性黑尾叶蝉群体的发病率一直保持在90.0%左右。低亲和性黑尾叶蝉群体的发病率维持在1%-4%。高亲和性群体在F7代的传毒率是87.7%,表明基本成功构建成一个高传毒的叶蝉群体,但是群体的高传毒特性只是在3个月无人工选择压力下的结果,在长时间内是否能保持这一搞得传毒特性还有待进一步的生物学验证。
运用mRNA差异显示技术筛选高、低亲和性黑尾叶蝉群体的差异基因,经差异显示,共筛选出差异条带42条,挑选差异显著、重扩增效果好的12条进行RNA斑点杂交验证,获得7个阳性片段,其中DD3、DD5、DD6、DD7和DD8等5个片段为高亲和性黑尾叶蝉群体所特有,DD2、DD11两个片段为低亲和性黑尾叶蝉群体特有。对阳性片段克隆、测序和同源性比较分析,揭示高亲和黑尾叶蝉群体所特有的DD5片段与人类20号染色体RP11-459I16克隆的DNA序列部分基因同源性高达99%;DD6片段与RDV的RNA2上的RNA结合蛋白部分基因同源性达94%,DD8片段序列与黑腹果蝇泛素部分基因有92%的同源性;DD3和DD7片段序列与已知序列同源性较低,推测是新的基因片段。DD2和DD11片段序列是低亲和性黑尾叶蝉群体特有,比对后发现与已知的序列同源性较低,同样推测是新的基因片段。
Rice dwarf virus (RDV) disease commonly occurred and even ran rampant in rice producing areas in the south of China. It has caused great decreases to rice yields in China. The virus is transmitted by Nephotettix cincticeps in a circulative, persistent manner. On our study, we constructed two groups,one is high affinity virus transmitter group and the other is a low affinity virus transmitter group. We use DDRT-PCR(differential display RT-PCR) to detection of the two groups’genes differentially expressed.
After artificially selection, two groups of Nephotettix cincticeps with contrasting virus- transmition efficiency were obtained. Biological assay indicated that the disease incidence rate of rice plants inoculated with the high virus- transmition Nephotettix cincticeps increased to 90.0%, compairing with the 1%- 4% of those inoculated with the low virus– transmition Nephotettix cincticeps. The trasmition rate of F7 derived from the high virus- transmition Nephotettix cincticeps was as high as 87.7% , demonstrating that a high virus- transmition Nephotettix cincticeps group was obtained. However, the high virus transmition competence was only a result of 3 months’selection wihout artificial selection pressure.Whether or not the group of the Nephotettix cincticeps could matain the competence needs further varification.
The high virus- transmition Nephotettix cincticeps and the low virus- transmition were investigated using DDRT-PCR for the difference gene expression. We cloned and sequenced seven Segment Segment DD5 belonged to the high- transmition Nephotettix cincticeps ,it was similar to human DNA sequence from clone RP11-459I16 on chromosome 20. Segment DD6 belonged to the high- transmition Nephotettix cincticeps it was similar to P2 protein of RDV. Segment DD8 belonged to the high- transmition Nephotettix cincticeps , it was similar to Drosophila melanogaster Ubiquitin-5E. Segment DD2, DD11, DD3 and DD7 was unknown. They can’t be find homologous genes when blasted, We presume they are new genes.
在构建高低亲和性黑尾叶蝉方面,无毒的黑尾叶蝉经过连续多代饲毒,传毒后,得到了对RDV传毒效率差异明显的两个黑尾叶蝉群体,通过生物学方法鉴定,高亲和性黑尾叶蝉群体的发病率一直保持在90.0%左右。低亲和性黑尾叶蝉群体的发病率维持在1%-4%。高亲和性群体在F7代的传毒率是87.7%,表明基本成功构建成一个高传毒的叶蝉群体,但是群体的高传毒特性只是在3个月无人工选择压力下的结果,在长时间内是否能保持这一搞得传毒特性还有待进一步的生物学验证。
运用mRNA差异显示技术筛选高、低亲和性黑尾叶蝉群体的差异基因,经差异显示,共筛选出差异条带42条,挑选差异显著、重扩增效果好的12条进行RNA斑点杂交验证,获得7个阳性片段,其中DD3、DD5、DD6、DD7和DD8等5个片段为高亲和性黑尾叶蝉群体所特有,DD2、DD11两个片段为低亲和性黑尾叶蝉群体特有。对阳性片段克隆、测序和同源性比较分析,揭示高亲和黑尾叶蝉群体所特有的DD5片段与人类20号染色体RP11-459I16克隆的DNA序列部分基因同源性高达99%;DD6片段与RDV的RNA2上的RNA结合蛋白部分基因同源性达94%,DD8片段序列与黑腹果蝇泛素部分基因有92%的同源性;DD3和DD7片段序列与已知序列同源性较低,推测是新的基因片段。DD2和DD11片段序列是低亲和性黑尾叶蝉群体特有,比对后发现与已知的序列同源性较低,同样推测是新的基因片段。
Rice dwarf virus (RDV) disease commonly occurred and even ran rampant in rice producing areas in the south of China. It has caused great decreases to rice yields in China. The virus is transmitted by Nephotettix cincticeps in a circulative, persistent manner. On our study, we constructed two groups,one is high affinity virus transmitter group and the other is a low affinity virus transmitter group. We use DDRT-PCR(differential display RT-PCR) to detection of the two groups’genes differentially expressed.
After artificially selection, two groups of Nephotettix cincticeps with contrasting virus- transmition efficiency were obtained. Biological assay indicated that the disease incidence rate of rice plants inoculated with the high virus- transmition Nephotettix cincticeps increased to 90.0%, compairing with the 1%- 4% of those inoculated with the low virus– transmition Nephotettix cincticeps. The trasmition rate of F7 derived from the high virus- transmition Nephotettix cincticeps was as high as 87.7% , demonstrating that a high virus- transmition Nephotettix cincticeps group was obtained. However, the high virus transmition competence was only a result of 3 months’selection wihout artificial selection pressure.Whether or not the group of the Nephotettix cincticeps could matain the competence needs further varification.
The high virus- transmition Nephotettix cincticeps and the low virus- transmition were investigated using DDRT-PCR for the difference gene expression. We cloned and sequenced seven Segment Segment DD5 belonged to the high- transmition Nephotettix cincticeps ,it was similar to human DNA sequence from clone RP11-459I16 on chromosome 20. Segment DD6 belonged to the high- transmition Nephotettix cincticeps it was similar to P2 protein of RDV. Segment DD8 belonged to the high- transmition Nephotettix cincticeps , it was similar to Drosophila melanogaster Ubiquitin-5E. Segment DD2, DD11, DD3 and DD7 was unknown. They can’t be find homologous genes when blasted, We presume they are new genes.
引文
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[2]方仲达.1997.植病研究方法,北京:农业出版社.
[3]王云华.2004.病毒晚期结构域研究进展.国外医学病毒学分册,11(1):13-16.
[4]叶楠,李永刚,文景芝.2008.利用mRNA差异显示技术(DDRT-PCR)研究大豆抗疫霉根腐病的分子机制.东北农业大学学报,39(9):6-9.
[5]曲志才,马向前,白逢伟,叶鸣明,潘重光,沈大棱.2002.活跃传毒介体灰飞虱(Laodelphax striatelllus)品系的杂交与选育.复旦学报(自然科学版),41(6):684-687.
[6]朱靖,杜立新.2005.反转录差异显示技术及其方法的改进.动物医学进展,26(1):19-21.
[7]阮义理,金登迪,陈光堉，林瑞芬,陈声祥.1981.水稻矮缩病的研究——Ⅰ.病史、病状和传播.植物保护学报,8(1):27-33.
[8]吴乃虎.1998.基因工程原理(第二版),北京:科学出版社,356-364.
[9]吴建国.2007.水稻矮缩病毒胁迫下的水稻基因表达谱分析(福建农林大学硕士学位论文),福州:福建农林大学.
[10]张维铭.2003.现代分子生物学试验手册,北京:科学出版社,419-422.
[11]李朝飞,庞义.2004.泛素-蛋白水解酶复合体通路与病毒侵染.生物工程学报,20(2):151-156.
[12]李毅,陈章良.2001.水稻病毒的分子生物学.科学出版社, 1～18.
[13]杨发青等.2004.白纹伊蚊感染等革2型病毒后基因表达变化的研究.寄生虫与医学昆虫学报,11(2):83-86.
[14]肖东来.2006.不同传毒能力灰飞虱群体的mRNA差异分析(福建农林大学硕士学位论文),福州:福建农林大学.
[15]洪健,李德宝等.2001.植物病毒分类图谱.科学出版社,59～70.
[16]崔莉.2005.不同寄生蜂寄生后小菜蛾精巢mRNA的表达差异.(河南农业大学硕士学位论文),郑州:河南农业大学.
[17]梁小波,鲁瑞芳,吴云锋,彭学贤.2001.植物病毒昆虫介体传播的研究进展.生物工程进展.
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