梨小食心虫滞育与非滞育幼虫抑制性消减文库的构建与分析
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摘要
梨小食心虫Grapholita molesta Busck属鳞翅目,小卷叶蛾科,是危害梨、桃等果树的重大害虫。本文分别以梨小食心虫滞育和非滞育幼虫cDNA为测试子和驱赶子,通过抑制性消减杂交技术,构建正反向差减cDNA文库,从中筛选梨小食心虫滞育相关基因,并对其进行测序和分析;通过RT-PCR技术筛选并克隆梨小食心虫持家基因,以期从分子水平上揭示梨小食心虫的滞育机理,为通过生态调控进行防治提供理论依据和新思路。主要结果如下:
1.通过抑制性消减杂交方法,构建了梨小食心虫滞育与非滞育幼虫正反向差减cDNA文库。经过建库过程各个环节的检测和对文库的测序分析表明,本实验过程中建立的两个差减文库质量较高,获得了128个滞育特异和132个非滞育特异的EST序列片段,通过进一步对检索结果进行分析,对不同差异序列中同源性较高,片段一致或交叉,以及可能来自同一基因的不同区域片段的基因合并,最后分别得到滞育差异表达EST序列42条(17条功能未知),非滞育差异表达EST序列46条(22条功能未知)。对得到的差异表达EST片段同源检索后推测,其功能大部分与滞育或非滞育相关。这些EST序列基本反映了梨小食心虫在滞育与非滞育期间的基因表达谱。为今后深入研究梨小食心虫滞育的分子机理奠定了基础。
2.在差减文库中筛选出了一些同源性较高的滞育与非滞育特征基因,在已知功能序列分析后发现,在滞育与非滞育文库中除转录相关基因比例相近外,在代谢酶、贮藏蛋白、信号传导和滞育关联蛋白基因含量均有较大的差异。发现在滞育个体中代谢酶和滞育关联蛋白基因表达量较高;而在非滞育个体中贮藏蛋白和信号传递基因表达量则较高。获得了芳基贮藏蛋白、14-3-3 Zeta-信号传导蛋白、富蛋氨酸贮藏蛋白、DEAD boxRNA解旋酶过氧化氢酶、热激蛋白90等与非滞育个体准备进入蛹期密切相关的基因和与滞育密切相关的苹果酸脱氢酶、滞育关联蛋白1、滞育关联蛋白2、保幼激素敏感血淋巴蛋白以及性信息素激活肽等。并对其进行了初步分析。
3.克隆了梨小食心虫微管蛋白和肌动蛋白等的持家基因,它们在滞育与非滞育幼虫体内表达稳定,说明这两个基因均可作为梨小食心虫持家基因使用。并筛选出没有RsaI酶切位点的肌动蛋白基因,适合用于检测SSH接头连接效率。
本研究中取得的结果可丰富对昆虫滞育分子机理的认识,也为今后进一步深入研究梨小食心虫滞育的分子机理打下了基础。
Grapholita molesta Busck (Lepidoptera: Olethreutidae) is a key pest seriously damaging fruit trees such as peach and pear. In this research, we constructed suppression subtractive hybridization cDNA library about diapause and non-diapause larvae of G. molesta to obtain the diapause-related genes which were then sequenced and analyzed, and we also cloned the housekeeping genes of G. molesta by RT-PCR. The paper aims to reveal the molecular mechanism of diapause and provide a new theory preventing and controlling the pest by methods of ecological regulation. The main results are as follows:
1. Two suppression subtractive hybridization cDNA libraries were constructed with diapause and non-diapause G. molesta as positive and reverse library. After identifying the quality of the libraries, the differential fragments of the two libraries were respectively selected for sequencing, of which 128 fragments were from the positive library, and 132 fragments were from the reverse library. After cluster analysis and ordering repeat sequences, finally 42 ESTs sequences with diapause difference and 46 ESTs sequences with non-diapause difference were obtained. After BLASTN, we found most of genes screened from two libraries are related to diapause and basically reflect gene expression profiles of G. molesta in duration of diapause and non-diapause. The results also established a foundation for further research of the molecular mechanism of diapauses of G. molesta.
2. Some high homology informative genes related to diapause or pupation were screened in this two SSH libraries. In addition to transcription related genes we also found metabolic enzyme and diapause related genes with higher content in positive library and storage protein and signal conduction genes are high expressed in reverse library. Malate dehydrogenase (MDH) gene, diapause associated protein 1 gene, diapause associated protein 2 gene, basic juvenile hormone sensitive hemolymph protein gene and pheromone biosynthesis activating neuropeptide (PBAN) gene etc. were found in positive library. And arylphorin-type storage protein gene, 14-3-3-zeta-protein gene, methionine-rich storage protein (SP2) gene, DEAD box RNA helicase gene, heat shock protein 90 gene which related to entering the pupal stage in reverse library.
3. Actin and tubulin gene of G. molesta were cloned by referring to the house-keeping genes of other Lepidoptera insects. It was found that the cloned actin and tubulin gene were both well expressed in different growth stages of G. molesta and one actin gene was not affected by RsaI digestion, thus it can be used to detect the efficiency of tester’s adaptor ligation in SSH.
The results of this research provide rich files of the molecular mechanism of insect diapause and found the bases for further molecular research in G. molesta as well.
1.通过抑制性消减杂交方法,构建了梨小食心虫滞育与非滞育幼虫正反向差减cDNA文库。经过建库过程各个环节的检测和对文库的测序分析表明,本实验过程中建立的两个差减文库质量较高,获得了128个滞育特异和132个非滞育特异的EST序列片段,通过进一步对检索结果进行分析,对不同差异序列中同源性较高,片段一致或交叉,以及可能来自同一基因的不同区域片段的基因合并,最后分别得到滞育差异表达EST序列42条(17条功能未知),非滞育差异表达EST序列46条(22条功能未知)。对得到的差异表达EST片段同源检索后推测,其功能大部分与滞育或非滞育相关。这些EST序列基本反映了梨小食心虫在滞育与非滞育期间的基因表达谱。为今后深入研究梨小食心虫滞育的分子机理奠定了基础。
2.在差减文库中筛选出了一些同源性较高的滞育与非滞育特征基因,在已知功能序列分析后发现,在滞育与非滞育文库中除转录相关基因比例相近外,在代谢酶、贮藏蛋白、信号传导和滞育关联蛋白基因含量均有较大的差异。发现在滞育个体中代谢酶和滞育关联蛋白基因表达量较高;而在非滞育个体中贮藏蛋白和信号传递基因表达量则较高。获得了芳基贮藏蛋白、14-3-3 Zeta-信号传导蛋白、富蛋氨酸贮藏蛋白、DEAD boxRNA解旋酶过氧化氢酶、热激蛋白90等与非滞育个体准备进入蛹期密切相关的基因和与滞育密切相关的苹果酸脱氢酶、滞育关联蛋白1、滞育关联蛋白2、保幼激素敏感血淋巴蛋白以及性信息素激活肽等。并对其进行了初步分析。
3.克隆了梨小食心虫微管蛋白和肌动蛋白等的持家基因,它们在滞育与非滞育幼虫体内表达稳定,说明这两个基因均可作为梨小食心虫持家基因使用。并筛选出没有RsaI酶切位点的肌动蛋白基因,适合用于检测SSH接头连接效率。
本研究中取得的结果可丰富对昆虫滞育分子机理的认识,也为今后进一步深入研究梨小食心虫滞育的分子机理打下了基础。
Grapholita molesta Busck (Lepidoptera: Olethreutidae) is a key pest seriously damaging fruit trees such as peach and pear. In this research, we constructed suppression subtractive hybridization cDNA library about diapause and non-diapause larvae of G. molesta to obtain the diapause-related genes which were then sequenced and analyzed, and we also cloned the housekeeping genes of G. molesta by RT-PCR. The paper aims to reveal the molecular mechanism of diapause and provide a new theory preventing and controlling the pest by methods of ecological regulation. The main results are as follows:
1. Two suppression subtractive hybridization cDNA libraries were constructed with diapause and non-diapause G. molesta as positive and reverse library. After identifying the quality of the libraries, the differential fragments of the two libraries were respectively selected for sequencing, of which 128 fragments were from the positive library, and 132 fragments were from the reverse library. After cluster analysis and ordering repeat sequences, finally 42 ESTs sequences with diapause difference and 46 ESTs sequences with non-diapause difference were obtained. After BLASTN, we found most of genes screened from two libraries are related to diapause and basically reflect gene expression profiles of G. molesta in duration of diapause and non-diapause. The results also established a foundation for further research of the molecular mechanism of diapauses of G. molesta.
2. Some high homology informative genes related to diapause or pupation were screened in this two SSH libraries. In addition to transcription related genes we also found metabolic enzyme and diapause related genes with higher content in positive library and storage protein and signal conduction genes are high expressed in reverse library. Malate dehydrogenase (MDH) gene, diapause associated protein 1 gene, diapause associated protein 2 gene, basic juvenile hormone sensitive hemolymph protein gene and pheromone biosynthesis activating neuropeptide (PBAN) gene etc. were found in positive library. And arylphorin-type storage protein gene, 14-3-3-zeta-protein gene, methionine-rich storage protein (SP2) gene, DEAD box RNA helicase gene, heat shock protein 90 gene which related to entering the pupal stage in reverse library.
3. Actin and tubulin gene of G. molesta were cloned by referring to the house-keeping genes of other Lepidoptera insects. It was found that the cloned actin and tubulin gene were both well expressed in different growth stages of G. molesta and one actin gene was not affected by RsaI digestion, thus it can be used to detect the efficiency of tester’s adaptor ligation in SSH.
The results of this research provide rich files of the molecular mechanism of insect diapause and found the bases for further molecular research in G. molesta as well.
引文
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冯恒林,于存周. 2006.砀山地区梨小食心虫的发生与防治.北方果树,(4):57
顾克余,翟虎渠. 1999.抑制性扣除杂交技术(SSH)及其在基因克隆上的研究进展.生物技术通报,(2):13～16
候宪春,王丹青,李体鹏. 2001.梨小食心虫的发生与综合防治.山东林业科技(增1):49
季相华. 2008.稻水象甲夏季滞育相关基因的分离与鉴定. [硕士学位论文].杭州:浙江大学
黎彦,杜华,段银昌. 2007. 2006年黄河故道地区梨小食心虫大发生的原因及防治对策.果农之友,(7):34
林露菲,刘依华,黄邦侃. 1998.福建桃、梨果蛀虫及梨小食心虫初步研究.华东昆虫学报, 7(2):35～40
刘红敏,汪新娥,胡肄珍. 2005.梨小食心虫的发生与防治.河南农业科学, (1):74～75
刘文荣,张木清. 2006.抑制性消减杂交技术在植物基因克隆中的应用.亚热带农业研究, 2(2):66～70
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