茶樹(shù)種質(zhì)資源發(fā)掘及新品種（系）選育

江昌俊　李葉云　韋朝領(lǐng)　梁名志　馬春雷

摘 要：該研究利用cDNA-AFLP技術(shù)分析茶樹(shù)被茶尺蠖取食誘導(dǎo)前后的基因表達(dá)譜差異，共計(jì)獲得231條差異條帶。以獲得的差異EST序列為基礎(chǔ)，成功克隆了乙醇脫氫酶、羥甲基丁烯基磷酸合成酶、羥甲基丁烯基磷酸還原酶、丁烯基磷酸還原酶等抗蟲(chóng)功能基因，并發(fā)現(xiàn)miRNA在茶樹(shù)被茶尺蠖取食誘導(dǎo)所產(chǎn)生的防御機(jī)制中具有重要的轉(zhuǎn)錄后調(diào)節(jié)作用。成功構(gòu)建了茶樹(shù)低溫脅迫SSH文庫(kù)，獲得了差異表達(dá)的UniESTs序列247條；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、抗壞血酸過(guò)氧化物酶、轉(zhuǎn)酮醇酶基因等抗寒功能基因，利用q-PCR技術(shù)分析了低溫脅迫下茶樹(shù)不同器官的表達(dá)水平。從茶樹(shù)中克隆出茶樹(shù)脫水素基因，獲得了轉(zhuǎn)基因煙草植株。成功構(gòu)建植物真核表達(dá)載體pBI121-CsCBF1，獲得轉(zhuǎn)基因煙草。在蛋白組學(xué)水平上系統(tǒng)的探討低溫脅迫下茶樹(shù)的抗性特征和代謝過(guò)程。克隆了茶樹(shù)谷氨酰胺合成酶基因、茶樹(shù)硝酸還原酶基因、AMP脫氨酶基因等氮代謝相關(guān)基因。開(kāi)發(fā)獲得了66條茶樹(shù)被茶尺蠖取食誘導(dǎo)后根部差異表達(dá)的EST序列、200余條與茶氨酸生物合成相關(guān)的EST。獲得了403個(gè)可用于茶樹(shù)遺傳作圖的SSR位點(diǎn)；通過(guò)cSNP相關(guān)性分析發(fā)現(xiàn)995位點(diǎn)與咖啡堿含量呈顯著相關(guān)。茶樹(shù)芽葉轉(zhuǎn)錄中共有7，457條unigenes在KEGG數(shù)據(jù)庫(kù)得到了注釋?zhuān)⑦M(jìn)一步歸類(lèi)到KEGG代謝通路的5個(gè)分支，針對(duì)次級(jí)代謝途徑進(jìn)行比對(duì)，發(fā)現(xiàn)了兒茶素、咖啡堿、茶氨酸相關(guān)氮代謝和萜類(lèi)代謝等途徑上的相關(guān)基因269個(gè)。建立了一套利用生理指標(biāo)評(píng)價(jià)茶樹(shù)資源抗寒性的體系。選育出綠茶、烏龍茶、紅茶和普洱茶新品種（系）38個(gè)。綠茶品種（系）19個(gè)、紅茶品種（系）4個(gè)、烏龍茶新品系13個(gè)、普洱茶新品系2個(gè)。選育出茶樹(shù)特異種質(zhì)32份。其中低咖啡堿株系11個(gè)、高氨基酸株系11個(gè)、高兒茶素株系9個(gè)。通過(guò)人工雜交和人工雜交結(jié)合理化誘變創(chuàng)制了900余份新材料。

關(guān)鍵詞：茶樹(shù) 種質(zhì)資源 功能基因 新品種 選育

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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