| Tea plant(Camellia sinensis) is a subtropical evergreen perennial plant, originated in southwest China, northern Burma and Indian Assam area. Tea plant is an important economic crop while tea is also the important drinks daily in life. The demand for tea has increased in recent years and tea planting region moved northward, but tea is a thermophilic plant that makes it hard for some of the Southern elite germplasms to move to Northern tea growing areas. Most of our tea-growing areas have suffered damage in winter and tea production declined dramatically as a result.Shaanxi Province is the northernmost growing area and has a long history of tea production. In the long reproduction and evolution, climate and environmental changes will certainly produce many unique qualities of tea germplasms and form the rich genetic diversity. Shaanxi tea-growing areas are part of Jiangbei tea-growing areas, which have a relatively high latitude and lower annual average temperature. Shaanxi local tea germplasms are very likely to form some morphological and physiological feature of the cold stress tolerance gradually. Therefore, it has a very importantly theoretical and practical significance for enhancing the yield and quality to research and evaluate the cold resistance of Shaanxi tea.The activities of superoxide dismutase (SOD) and catalase (CAT), the soluble protein content and the electric conductivity at a low temperature of29tea germplasm resources were detected to make a comprehensive evaluation of the cold resistance of Shaanxi tea germplasm; Gene expression differences during cold stress of tea plant were preliminary studied by mRNA differential display reverse transcription PCR(DDRT-PCR) with the Shaanxi tea plant with the excellent cold resistance germplasm. Meanwhile, the expressional characteristics of differentially expressional gene are studied by semi-quantitative RT-PCR. We gained these results:1. Four physiological and biochemical indices are determined to do a comprehensive evaluation of the cold resistance of Shaanxi tea germplasm. The results showed that the evaluation value D of29tea germplasms range from0.201to0.933. The D value of Xixiangdahe6is maximum, indicating that it has the strongest cold resistance; Sichuannanjiang variety’s D value is the minimum of only0.201, belongs to low freeze resistance group.2. Based on the cluster analysis, the29tea cultivars were divided into three groups, namely high, medium, and low cold resistance cultivar group. Xixiangdahe6, Ziyangyuanye, Xixiangdahe8, Shubei variety of Japan, Pinglisanyang and longjing43belong to high cold resistance group; The low cold resistance group covers Xixiangdahe10, Ziyangdayepao, Fudingdabaicha, Sichuannanjiang variety; the others cultivars belong to medium cold resistance group.3. The SOD, CAT activity of Xixiangdahe6is higher. Ziyangyuanye has a higher content of soluble protein and Xixiangdahe8has a lower electrolyte leakage rate after-10℃subzero treatment. The above three tea varieties have the strongest cold resistance for all the tea germplasm tested, which are native to Qinba mountain area in Shaanxi Province while the introduced variety Fudingdabaicha’D values minimum. Combined with related morphological and anatomical results, part of Shaanxi Province unique tea resources have more excellent cold resistance than the introduced variety.4. Gene expression differences during cold stress is studied by DDRT-PCR with Ziyangyuanye and three cDNA fragments related to cold resistance were identified, they are DF-2(B0329/B0305), DF-3(B0329/B0305)and DF-8(B0329/B0311).5. It is obtained by sequencing and homology comparison analysis that Csgsf (DF-8) is the glutamine synthetase gene fragment of tea and Semi-quantitative RT-PCR analysis showed that Csgsf expression gradually decreased during cold stress; Cscaf2(DF-3) is the gene fragment to responsed to drought and low temperature; Cscafl is281bp, whose expression level decreased gradually with the cold stress.6. The modified SDS/Phenol is established to gain high purity and integrity total RNA from tea plant. Adding insoluble PVPP when grinding and joining PVP and β-mercaptoethanol can make polyphenols not easy to be oxidized. Adding1/3volume NaAc to supernatant is an effective method for polysaccharide precipitation. Following this efficient procedure, we routinely obtained the total RNA with high quality and good integrity. The ratio of A260/A280is1.9-2.0. The tolal RNA is highly stable and sufficiently pure for DDRT-PCR.7. Four physical and chemical indicators are combined into two independent indicators in tea cold resistance comprehensive evaluation using principal component analysis. Combined with the membership function values method, the common method in plant resistance, we obtained the D value, which is a dimensionless pure number that makes it comparable among different tea varieties. The results showed that it can reveal the relationship between the indicators and the cold resistance by selecting several physiological and biochemical indicators and using principal component analysis and the membership function value method. |
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