茶树种质资源发掘及新品种（系）选育

江昌俊　李叶云　韦朝领　梁名志　马春雷

摘 要：该研究利用cDNA-AFLP技术分析茶树被茶尺蠖取食诱导前后的基因表达谱差异，共计获得231条差异条带。以获得的差异EST序列为基础，成功克隆了乙醇脱氢酶、羟甲基丁烯基磷酸合成酶、羟甲基丁烯基磷酸还原酶、丁烯基磷酸还原酶等抗虫功能基因，并发现miRNA在茶树被茶尺蠖取食诱导所产生的防御机制中具有重要的转录后调节作用。成功构建了茶树低温胁迫SSH文库，获得了差异表达的UniESTs序列247条；克隆了蔗糖磷酸合成酶、海藻糖-6-磷酸合成酶和α-葡萄糖苷酶、抗坏血酸过氧化物酶、转酮醇酶基因等抗寒功能基因，利用q-PCR技术分析了低温胁迫下茶树不同器官的表达水平。从茶树中克隆出茶树脱水素基因，获得了转基因烟草植株。成功构建植物真核表达载体pBI121-CsCBF1，获得转基因烟草。在蛋白组学水平上系统的探讨低温胁迫下茶树的抗性特征和代谢过程。克隆了茶树谷氨酰胺合成酶基因、茶树硝酸还原酶基因、AMP脱氨酶基因等氮代谢相关基因。开发获得了66条茶树被茶尺蠖取食诱导后根部差异表达的EST序列、200余条与茶氨酸生物合成相关的EST。获得了403个可用于茶树遗传作图的SSR位点；通过cSNP相关性分析发现995位点与咖啡碱含量呈显著相关。茶树芽叶转录中共有7，457条unigenes在KEGG数据库得到了注释，并进一步归类到KEGG代谢通路的5个分支，针对次级代谢途径进行比对，发现了儿茶素、咖啡碱、茶氨酸相关氮代谢和萜类代谢等途径上的相关基因269个。建立了一套利用生理指标评价茶树资源抗寒性的体系。选育出绿茶、乌龙茶、红茶和普洱茶新品种（系）38个。绿茶品种（系）19个、红茶品种（系）4个、乌龙茶新品系13个、普洱茶新品系2个。选育出茶树特异种质32份。其中低咖啡碱株系11个、高氨基酸株系11个、高儿茶素株系9个。通过人工杂交和人工杂交结合理化诱变创制了900余份新材料。

关键词：茶树 种质资源 功能基因 新品种 选育

Abstract： 231 differential bands were obtained on the gene expression profiles expressed after Ectropis oblique feeding. Alcoholde hydrogenase， hydroxymethylbutenyl diphosphate synthase， hydroxymethylbutenyl diphosphate reductase and butenyl diphosphate reductase insect resistant genes were successfully cloned. Meanwhile， it was found that miRNA has the important post-transcriptional regulation function in the defense mechanism induced by Ectropis oblique feeding. Tea SSH library of cold stress was generated， and 247 UniESTs with the differential expressions were finally acquired after spot blotting screening technology. Sucrose phosphate synthase， trehalose-6-phosphate synthase， α-glucosidase， ascorbate peroxidase and transketolase cold resistant functional genes were cloned. Dehydrin gene CsDHN1 was cloned from tea plant and over-expression on Nicotiana tabacum plants were generated. pBI121-CsCBF1 transgenetic Nicotiana tabacum plants were acquired. We also discussed tea resistant characteristics and metabolic process under cold stress on the level of proteomics， and glutamine synthetase， nitrate reductase， AMP deaminase genes which were related to nitrogen metabolism in tea plants were finally cloned. 200 EST sequences which were related to theanine biosynthesis were acquired. 403 SSR sites which could be used to construct the tea genetic map were acquired. The SNP site 995 was closely related to tea caffeine content after correlation analysis. 7，457 unigenes in the transcriptome of tea bud and leaf in KEGG database were annotated， and further， classified into 5 branches in KEGG metabolic pathway. Aiming at the secondary metabolic pathway， 269 genes were found to be related to catechins， caffeine and theanine metabolism and terpene metabolism. A tea cold resistance evaluation system was constructed by using some physiological indicator. 38 new tea cultivars were bred which are suitable to process green tea， oolong tea and black tea. And 19 belong to green tea cultivars. Four cultivars belong to black tea. 13 cultivars belong to oolong tea. Two cultivars belong to puer new tea. 32 distinctive tea germplasm were bred. Among them， 11 strains with low caffeine content， 11 strains with high amino acids content， one new strain with high amino acid content， 9 strains with high catechins content.More than 900 new plant materials were created by artificial hybridization and artificial hybridization combined with physiochemical mutagenesis.

Key Words： Tea plant （Camellia sinensis）； Germplasm resources； Functional gene； New cultivar； Breeding

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