基于SSH技术研究茶树被茶尺蠖取食诱导的基因差异表达
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摘要
茶树虫害一直是我国茶叶生产中优质和高效的主要限制因子,而茶树对害虫取食后所诱发的防御反应分子机制至今不清;同时常绿木本植物对害虫取食诱导防御的分子机理研究相当缺乏。本论文利用抑制性消减杂交(SSH)技术,在茶树叶片被主要害虫-茶尺蠖取食后,对茶叶中诱导表达的基因进行了筛选和研究,在此基础上通过荧光定量PCR技术,比较研究了与重要防御功能相关的差异表达片段(TDF)的表达特征和在不同品种间的表达变化差异。主要结果如下:
(1)建立了稳定可靠的SSH技术体系,为后续研究不同样本之间的基因差异表达提供了技术保障。
(2)通过对正向差减文库中包含的诱导表达cDNA片段进行克隆鉴定,并对长度大于200bp的片段进行测序,最终获得了280个TDF的序列。按照长度大小进行分类:长度在200bp-400bp之间的TDF有151个,占总量的54%;长度在400bp-600bp之间的TDF有101个,占总量的36%;长度大于600bp的TDF有28个,占总量的10%;通过Blastx比对显示:与已知功能基因序列具有较高同源性的的TDF有157个,占总量的56%;为推测蛋白的克隆子有19个,占总量的6%;没有任何匹配的有104个,占总量的37%。并已将其中的271个TDF序列登录在GenBank中(GW342615 - GW342885)。
(3)对其中144个诱导表达基因的推测功能进行分类得出的结果表明:可能与蛋白质代谢相关的基因有31个,占总体的21%;与光合作用和初级代谢功能相关的有18个,占总体的13%;与转运类蛋白功能相关的有11个,占总体的8%;与转录功能相关的有12个,占总体的8%;与信号转导功能相关的有11个,占总体的8%;与脂类代谢和次级代谢功能相关的分别有7个和13个,分别占总体的5%和9%;与逆境与防御功能相关的有22个,占总量的15%;与植物体其他代谢途径相关的有19个,占总量的13%。(4)依据现有相关植物对害虫防御机制的报道分析得出茶树被茶尺蠖取食诱导可能所涉及的防御途径:与直接防御相关的有4个TDF,分别涉及咖啡碱合成酶(TDF-55、88)、黄酮醇合成酶(TDF-72、146)、精氨酸酶(TDF-2)和半胱氨酸酶抑制剂(TDF-144);与间接防御相关的有7个TDF,分别涉及绿叶性挥发物、芳香族挥发物和萜烯类挥发物生物很成相关。
(5)通过定量PCR对诱导表达基因的表达量的分析,我们发现茶树受到茶尺蠖取食危害后,与不同防御机制相关的TDF上调表达量是不同的,且其随着时间的变化规律和在不同品种间表达量也存在差异。例如咖啡碱合成酶(TDF-55、88)和黄酮合成酶(TDF-72、146)受茶尺蠖取食后不同的时间里,二者都随着时间的增加而出现增加的现象,但是咖啡碱合成酶在取食后4小时增加的幅度明显比黄酮合成酶大。不同茶树品种被茶尺蠖取食诱导后,其诱导的差异片段的上调表达量也有不同。例如其中龙井43被取食后,TDF-73上调表达倍数比舒茶早高出最多,达到74.5%,TDF-55和TDF-139次之,分别比舒茶早高出57.8%和57.1%,TDF-40仅比舒茶早高出11.9%。而对于TDF-72和105来说则是龙井43的上调表达倍数比舒茶早低,分别为53.4%和26.9%。
Tea pests have always been the major limiting factor in high quality and efficiency tea production, however, the molecular mechanism of the tea plant resistance is not clear today. While the molecular mechanism induced defense research of feeding pests on evergreen woody plant is lacking. This paper researched the differentially expressed genes in both number and type after feeding main pests (tea looper) on the tea leaves using SSH technology. On this basis, comparative studies of the express characteristics of differentially expressed fragments(TDF)related with the important defense function, as well as the expression differences in different varieties were made through fluorescence quantitative PCR. Main results are as follows:
(1) A stable and reliable technical system of SSH is established, and technical support for subsequent study of differences in gene expression between different samples is provided.
(2) Based on the cloning identification of induced cDNA fragments included in the forward subtractive cDNA library, and the sequencing of the fragments longer than 200bp, 280 TDF sequences were obtained. According to the length, there are 151 TDF with the length between 200bp and 400bp, 54% of the total. The number of the TDF with the length between 400bp and 600bp is 101, 36% of the total. 28 TDF is longer than 600bp, 10% of the total. Through BlastX comparison shows: the gene has high homology with known functional sequence is 157, 56% of the total, the cloning of hypothetical protein is 19, 6% of the total. There's no match for 104, 37 % of the total. And 271 sequences of TDF is submitted to GenBank(GW342615 - GW342885)。
(3) The classification results of 144 induced genes’probable function show that 31 genes related to the protein metabolism, 21% of the total;18 have relationships with photosynthesis and primary metabolic functions, about 13%;11 are with the transfer function of proteins, 8% of the total; and the number of genes related to the transcription function is 12, 8% of the total;11 related to the function of signal transduction, 8% of the total;and the number of the genes related to lipid metabolism and secondary metabolic function are respectively 7 and 13, 5% and 9%;22 are related to adversity and defense functions, 15% of the total;19 are related to other metabolic pathways of plant, 13% of the total.
(4) According to the reports related to plant defense mechanism of pests, there are a few may involved in the tea looper feeding: 4 TDF related with direct defense, including caffeine synthase (TDF - 55, 88), flavonols synthase (TDF - 72, 146), arginine enzyme (TDF - 2) and homocysteine enzyme inhibitors (TDF - 144), And 7 TDF have relationships with indirect defense, including the processes of biosynthesis of green leafy volatiles, aromatic volatiles and terpenes volatiles.
(5) Through quantitative polymerase chain reaction (PCR) analysis of induced gene expression level, it is founded that after feeding,the raised expression of the TDF related to different defense mechanisms is different, and along with the time expression varies in different varieties. Such as the phenomenon that caffeine synthase (TDF - 55, 88) and flavonoids synthase (TDF - 72, 146) increase in different time after feeding, but caffeine synthase was increased significantly greater than flavonoids synthase in four hours after feeding. Different varieties of tea, the raised expression of the induced TDF is different, too. For example, after feeding, longjing 43, TDF - 73 raised express multiple higher than shuchazao, reach to 74.5%, then are the TDF - 55 and TDF - 139, higher than the shuchazao 57.8% and 57.1% ,TDF - 40, higher than shuchazao only 11.9%. For TDF - 72 and 105 are lower than shuchazao, respectively 53.4% and 26.9%.
(1)建立了稳定可靠的SSH技术体系,为后续研究不同样本之间的基因差异表达提供了技术保障。
(2)通过对正向差减文库中包含的诱导表达cDNA片段进行克隆鉴定,并对长度大于200bp的片段进行测序,最终获得了280个TDF的序列。按照长度大小进行分类:长度在200bp-400bp之间的TDF有151个,占总量的54%;长度在400bp-600bp之间的TDF有101个,占总量的36%;长度大于600bp的TDF有28个,占总量的10%;通过Blastx比对显示:与已知功能基因序列具有较高同源性的的TDF有157个,占总量的56%;为推测蛋白的克隆子有19个,占总量的6%;没有任何匹配的有104个,占总量的37%。并已将其中的271个TDF序列登录在GenBank中(GW342615 - GW342885)。
(3)对其中144个诱导表达基因的推测功能进行分类得出的结果表明:可能与蛋白质代谢相关的基因有31个,占总体的21%;与光合作用和初级代谢功能相关的有18个,占总体的13%;与转运类蛋白功能相关的有11个,占总体的8%;与转录功能相关的有12个,占总体的8%;与信号转导功能相关的有11个,占总体的8%;与脂类代谢和次级代谢功能相关的分别有7个和13个,分别占总体的5%和9%;与逆境与防御功能相关的有22个,占总量的15%;与植物体其他代谢途径相关的有19个,占总量的13%。(4)依据现有相关植物对害虫防御机制的报道分析得出茶树被茶尺蠖取食诱导可能所涉及的防御途径:与直接防御相关的有4个TDF,分别涉及咖啡碱合成酶(TDF-55、88)、黄酮醇合成酶(TDF-72、146)、精氨酸酶(TDF-2)和半胱氨酸酶抑制剂(TDF-144);与间接防御相关的有7个TDF,分别涉及绿叶性挥发物、芳香族挥发物和萜烯类挥发物生物很成相关。
(5)通过定量PCR对诱导表达基因的表达量的分析,我们发现茶树受到茶尺蠖取食危害后,与不同防御机制相关的TDF上调表达量是不同的,且其随着时间的变化规律和在不同品种间表达量也存在差异。例如咖啡碱合成酶(TDF-55、88)和黄酮合成酶(TDF-72、146)受茶尺蠖取食后不同的时间里,二者都随着时间的增加而出现增加的现象,但是咖啡碱合成酶在取食后4小时增加的幅度明显比黄酮合成酶大。不同茶树品种被茶尺蠖取食诱导后,其诱导的差异片段的上调表达量也有不同。例如其中龙井43被取食后,TDF-73上调表达倍数比舒茶早高出最多,达到74.5%,TDF-55和TDF-139次之,分别比舒茶早高出57.8%和57.1%,TDF-40仅比舒茶早高出11.9%。而对于TDF-72和105来说则是龙井43的上调表达倍数比舒茶早低,分别为53.4%和26.9%。
Tea pests have always been the major limiting factor in high quality and efficiency tea production, however, the molecular mechanism of the tea plant resistance is not clear today. While the molecular mechanism induced defense research of feeding pests on evergreen woody plant is lacking. This paper researched the differentially expressed genes in both number and type after feeding main pests (tea looper) on the tea leaves using SSH technology. On this basis, comparative studies of the express characteristics of differentially expressed fragments(TDF)related with the important defense function, as well as the expression differences in different varieties were made through fluorescence quantitative PCR. Main results are as follows:
(1) A stable and reliable technical system of SSH is established, and technical support for subsequent study of differences in gene expression between different samples is provided.
(2) Based on the cloning identification of induced cDNA fragments included in the forward subtractive cDNA library, and the sequencing of the fragments longer than 200bp, 280 TDF sequences were obtained. According to the length, there are 151 TDF with the length between 200bp and 400bp, 54% of the total. The number of the TDF with the length between 400bp and 600bp is 101, 36% of the total. 28 TDF is longer than 600bp, 10% of the total. Through BlastX comparison shows: the gene has high homology with known functional sequence is 157, 56% of the total, the cloning of hypothetical protein is 19, 6% of the total. There's no match for 104, 37 % of the total. And 271 sequences of TDF is submitted to GenBank(GW342615 - GW342885)。
(3) The classification results of 144 induced genes’probable function show that 31 genes related to the protein metabolism, 21% of the total;18 have relationships with photosynthesis and primary metabolic functions, about 13%;11 are with the transfer function of proteins, 8% of the total; and the number of genes related to the transcription function is 12, 8% of the total;11 related to the function of signal transduction, 8% of the total;and the number of the genes related to lipid metabolism and secondary metabolic function are respectively 7 and 13, 5% and 9%;22 are related to adversity and defense functions, 15% of the total;19 are related to other metabolic pathways of plant, 13% of the total.
(4) According to the reports related to plant defense mechanism of pests, there are a few may involved in the tea looper feeding: 4 TDF related with direct defense, including caffeine synthase (TDF - 55, 88), flavonols synthase (TDF - 72, 146), arginine enzyme (TDF - 2) and homocysteine enzyme inhibitors (TDF - 144), And 7 TDF have relationships with indirect defense, including the processes of biosynthesis of green leafy volatiles, aromatic volatiles and terpenes volatiles.
(5) Through quantitative polymerase chain reaction (PCR) analysis of induced gene expression level, it is founded that after feeding,the raised expression of the TDF related to different defense mechanisms is different, and along with the time expression varies in different varieties. Such as the phenomenon that caffeine synthase (TDF - 55, 88) and flavonoids synthase (TDF - 72, 146) increase in different time after feeding, but caffeine synthase was increased significantly greater than flavonoids synthase in four hours after feeding. Different varieties of tea, the raised expression of the induced TDF is different, too. For example, after feeding, longjing 43, TDF - 73 raised express multiple higher than shuchazao, reach to 74.5%, then are the TDF - 55 and TDF - 139, higher than the shuchazao 57.8% and 57.1% ,TDF - 40, higher than shuchazao only 11.9%. For TDF - 72 and 105 are lower than shuchazao, respectively 53.4% and 26.9%.
引文
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