茶树次黄嘌呤核苷酸脱氢酶基因克隆及其与咖啡碱含量的关联分析
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摘要
咖啡碱是嘌呤碱中最重要的一种，是茶树中的次级代谢产物，对茶叶的滋味起重要的作用，对于咖啡碱代谢途径的研究有重要的意义。目前，代谢途径中核心途径的关键酶基因已经被克隆，而供体途径中的关键酶次黄嘌呤核苷酸脱氢酶（IMPDH）只有一条cDNA被克隆。因此，获得次黄嘌呤核苷酸脱氢酶的完整基因信息，并分析其与咖啡碱含量的关系，可以为茶树咖啡碱含量的遗传改良打下理论基础。本文的主要研究结果如下：
1．根据NCBI登录的TIDH (EU106658)基因（编码次黄嘌呤核苷酸脱氢酶）cDNA设计引物，克隆获得了5条基因组DNA的中间片段，然后获得TIDH4全长DNA序列；并克隆了3条新的cDNA，它们之间差异很小，其中一条开放阅读框长1,335bp，编码444个氨基酸，推测分子量为46.7KD，理论等电点值为6.62，进化树分析表明茶树TIDH基因cDNA与葡萄亲缘关系最近。
将基因组DNA与cDNA进行比对，结果显示TIDH1和TIDH2分别与cDNA2-13和2-15完全匹配，两个基因片段都含有4个外显子和3个内含子，推测TIDH1和TIDH2两个基因片段在茶树中是表达的。
2．选择TIDH3基因分析核苷酸多样性，在336~1094bp区域共发现48个SNP位点，其中转换28个，包括A-G15个和C-T13个；颠换20个，包括A-C7个、A-T3个、C-G6个和T-G4个。
总核苷酸多样性指数π和θ分别为0.006和0.012。同义突变多样性πsyn=0.00932大于非同义突变多样性πnonsyn=0.00609，说明TIDH基因的同义突变的遗传变异程度大于非同义突变。Ka/Ks=0.5962＜1，认为TIDH基因受到负向选择作用。对TIDH基因作连锁不平衡分析，当显著性值P<0.05时，有20.8%（25个）的位点间存在显著的连锁不平衡。当采用更严格的P<0.001时，则位点间显著连锁不平衡数降低为20个，占总数的16.7%。
从前期建立的中国茶树资源核心种质中选取咖啡碱含量有代表性的95份资源进行了二个年度、春秋二季的咖啡碱含量HPLC测定结果表明，大部分材料的咖啡碱含量在2.5%~4.5%,,四次重复的平均值为3.5%。变异系数在15%~19%，说明茶叶咖啡碱含量在不同年份与季节都是比较稳定。
基于候选基因策略的关联分析结果显示，当考虑群体结构时，找到两个SNP位点与咖啡碱含量显著相关，位点效应值为10.43%和6.40%。
Caffeine is one of the most important purine alkaloids and they are secondary metabolites in teaplant, as they play an important role in flavour of tea. It’s of significance to study the metabolicpathway of caffeine. Now, the key enzymes of core pathway have already been cloned, while only onecDNA of IMP dehydrogenase (IMPDH: the key enzyme) was cloned in the provider pathway. Sogetting the full gene information of IMPDH and analysing the gene’s relationship with caffeinecontent will lay a foundation for genetic improvement of caffeine content in tea. The main results areas follow:
1. The primers were designed based on TIDH (which codes for IMPDH) cDNA (EU106658)published in the NCBI. Five internal fragments of genomic DNA were cloned. After that, the fulllength DNA of TIDH4was cloned.Meanwhile, three new cDNA were cloned. As there is littledifference between the3cDNAs, we select one to analyse its bioinformatic information. It has a1,335bp coding region, encoding444amino acids with the putative molecular weight of46.7KD andtheoretical isoelectric point6.62. The tea plant has much closer relationship with Vitis vinifera in thephylogenetic analysis of the TIDH cDNA.
When alignment between DNA and cDNA, TIDH1matched with cDNA2-13and TIDH2withcDNA2-15, and both DNA had4exons and3introns. So it is considered that TIDH1and TIDH2areexpressed in tea plant.
2. TIDH3was selected to analyse nucleotide diversity,48SNPs were found in336~1094bpregion, including28transitions (15A-G and13C-T),20transversions(7A-C,3A-T,6C-G and4T-G).
The nucleotide diversityπ=0.006and θ=0.012, respectively. The diversity at synonymous sites,πsyn=0.00932, was larger than diversity at non-synonymous sites, πnonsyn=0.00609, that indicated thegenetic variation level of synonymous mutation is larger than that of non-synonymous mutation.Ka/Ks=0.5962was less than one, indicating the action of purifying selection. The linkagedisequilibrium of SNPs was detected, when the significance value was lower than0.05, there were20.8%sites being significantly linkage disequilibrium; when the significance value was lower than0.001, the number of that dropped to16.7%.
A set of95accession tea genetic resources which are typical in caffeine content was selectedfrom core collection of Chinese tea germplasm established before. The caffeine content of twoseasons of spring and autumn in two years was tested by HPLC method. The result showed: thecaffeine content of most germplasms was2.5%~4.5%, the average content of four duplicates was3.5%. The CV is15%~19%, which means the caffeine content in tea is stable in differerent years andreasons.
The results of association analysis based on candidate genes showed that, when the result ofpopulation structure was considered, two SNPs were found to be significantly correlated with caffeine content by association analysis, their effect value were10.43%and6.40%respectively.
1．根据NCBI登录的TIDH (EU106658)基因（编码次黄嘌呤核苷酸脱氢酶）cDNA设计引物，克隆获得了5条基因组DNA的中间片段，然后获得TIDH4全长DNA序列；并克隆了3条新的cDNA，它们之间差异很小，其中一条开放阅读框长1,335bp，编码444个氨基酸，推测分子量为46.7KD，理论等电点值为6.62，进化树分析表明茶树TIDH基因cDNA与葡萄亲缘关系最近。
将基因组DNA与cDNA进行比对，结果显示TIDH1和TIDH2分别与cDNA2-13和2-15完全匹配，两个基因片段都含有4个外显子和3个内含子，推测TIDH1和TIDH2两个基因片段在茶树中是表达的。
2．选择TIDH3基因分析核苷酸多样性，在336~1094bp区域共发现48个SNP位点，其中转换28个，包括A-G15个和C-T13个；颠换20个，包括A-C7个、A-T3个、C-G6个和T-G4个。
总核苷酸多样性指数π和θ分别为0.006和0.012。同义突变多样性πsyn=0.00932大于非同义突变多样性πnonsyn=0.00609，说明TIDH基因的同义突变的遗传变异程度大于非同义突变。Ka/Ks=0.5962＜1，认为TIDH基因受到负向选择作用。对TIDH基因作连锁不平衡分析，当显著性值P<0.05时，有20.8%（25个）的位点间存在显著的连锁不平衡。当采用更严格的P<0.001时，则位点间显著连锁不平衡数降低为20个，占总数的16.7%。
从前期建立的中国茶树资源核心种质中选取咖啡碱含量有代表性的95份资源进行了二个年度、春秋二季的咖啡碱含量HPLC测定结果表明，大部分材料的咖啡碱含量在2.5%~4.5%,,四次重复的平均值为3.5%。变异系数在15%~19%，说明茶叶咖啡碱含量在不同年份与季节都是比较稳定。
基于候选基因策略的关联分析结果显示，当考虑群体结构时，找到两个SNP位点与咖啡碱含量显著相关，位点效应值为10.43%和6.40%。
Caffeine is one of the most important purine alkaloids and they are secondary metabolites in teaplant, as they play an important role in flavour of tea. It’s of significance to study the metabolicpathway of caffeine. Now, the key enzymes of core pathway have already been cloned, while only onecDNA of IMP dehydrogenase (IMPDH: the key enzyme) was cloned in the provider pathway. Sogetting the full gene information of IMPDH and analysing the gene’s relationship with caffeinecontent will lay a foundation for genetic improvement of caffeine content in tea. The main results areas follow:
1. The primers were designed based on TIDH (which codes for IMPDH) cDNA (EU106658)published in the NCBI. Five internal fragments of genomic DNA were cloned. After that, the fulllength DNA of TIDH4was cloned.Meanwhile, three new cDNA were cloned. As there is littledifference between the3cDNAs, we select one to analyse its bioinformatic information. It has a1,335bp coding region, encoding444amino acids with the putative molecular weight of46.7KD andtheoretical isoelectric point6.62. The tea plant has much closer relationship with Vitis vinifera in thephylogenetic analysis of the TIDH cDNA.
When alignment between DNA and cDNA, TIDH1matched with cDNA2-13and TIDH2withcDNA2-15, and both DNA had4exons and3introns. So it is considered that TIDH1and TIDH2areexpressed in tea plant.
2. TIDH3was selected to analyse nucleotide diversity,48SNPs were found in336~1094bpregion, including28transitions (15A-G and13C-T),20transversions(7A-C,3A-T,6C-G and4T-G).
The nucleotide diversityπ=0.006and θ=0.012, respectively. The diversity at synonymous sites,πsyn=0.00932, was larger than diversity at non-synonymous sites, πnonsyn=0.00609, that indicated thegenetic variation level of synonymous mutation is larger than that of non-synonymous mutation.Ka/Ks=0.5962was less than one, indicating the action of purifying selection. The linkagedisequilibrium of SNPs was detected, when the significance value was lower than0.05, there were20.8%sites being significantly linkage disequilibrium; when the significance value was lower than0.001, the number of that dropped to16.7%.
A set of95accession tea genetic resources which are typical in caffeine content was selectedfrom core collection of Chinese tea germplasm established before. The caffeine content of twoseasons of spring and autumn in two years was tested by HPLC method. The result showed: thecaffeine content of most germplasms was2.5%~4.5%, the average content of four duplicates was3.5%. The CV is15%~19%, which means the caffeine content in tea is stable in differerent years andreasons.
The results of association analysis based on candidate genes showed that, when the result ofpopulation structure was considered, two SNPs were found to be significantly correlated with caffeine content by association analysis, their effect value were10.43%and6.40%respectively.
引文
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