【作者】 张莹；

【导师】 唐茜；

【作者基本信息】 四川农业大学 ， 茶学， 2021， 博士

【摘要】 四川是茶树发源地之一,茶树种质资源十分丰富,为培育优质特色茶树新品种奠定了坚实的基础。崇州枇杷茶是经过长期自然演化和半人工驯化形成的一个四川地方茶树群体品种资源,具有生长势旺、叶大似枇杷叶、适应性和耐寒性强等特点。近二十年来,因受到自然灾害、人为砍伐、过度开发等因素影响,野生崇州枇杷茶数量锐减,为更好保护与开发利用该地方资源,课题组历时十余年采用系统选育法选育出26个崇州枇杷茶新品系,本研究对崇州枇杷茶新品系形态特征、生化特性、光合性能、抗寒特性进行了多元评价,并采用高通量转录组测序技术和代谢组学技术评价抗寒性的分子调控和代谢应答机理,旨在深入了解崇州枇杷茶资源的遗传多样性、亲缘关系、生理生化特性与抗寒机制,以期为崇州枇杷茶资源的开发利用以及特异优茶树资源的筛选和新品种的选育提供理论基础。研究结果如下:1.26个崇州枇杷茶新品系的表型性状具有从野生型向栽培型演化的特点,如叶形椭圆、叶尖渐尖等,但仍有部分品系具叶大花大、叶齿稀钝、叶面隆起、子房无茸毛等野生型茶树性状;大部分品系处于为过渡型。供试品系表型性状变异系数均值为34.17%,多样性指数为0.70,基于SSR标记的多态信息含量为0.636,显示崇枇新品系表型及遗传多样性水平较高,其表型形态稳定性为芽叶>叶片>树体>花,一芽三叶百芽重、叶长、叶宽等产量关联性状的遗传分化水平较高,其中崇枇16号、20号、23号、24号、26号生长势强,具有选育高产茶树品种的潜力;崇枇3号、5号、11号、20号芽叶颜色为紫色,具有选育高花青素含量茶树品种的潜力;崇枇2号、3号、5号、6号等11个品系一芽一叶期比对照品种福鼎大白早6～15天,具有选育特早生茶树品种的潜力。2.连续3年的供试新品系的主要生化成分检测结果表明,春夏秋三季新梢的茶多酚含量为16.76%～28.28%,其中崇枇3号、11号、23号在三季的含量均>25%,为高茶多酚特异茶树品系;新梢氨基酸含量为1.74%～5.69%,其中崇枇25号、26号春季新梢氨基酸总量分别为5.69%、5.18%,为高氨基酸茶树品系;新梢咖啡碱平均含量为4.10%～4.48%,其中崇枇2号、10号含量分别为5.03%、5.29%,符合高咖啡碱茶树种质资源标准;新梢儿茶素组分变异系数为0.86%～22.35%,其中非酯型儿茶素变异系数为2.16%～7.82%,酯型儿茶素变异系数为8.81%～19.16%,崇枇11号、12号、22号、23号三季的儿茶素总量均值分别为23.04%、21.69%、23.48%和23.90%,属于高儿茶素含量品系;崇枇11号、12号、22号、23号的EGCG含量在三季的均值分别为16.25%、15.31%、15.92%和15.95%,属于高EGCG茶树品系。3.对崇枇新品系的抗寒性进行田间直接鉴定、基于抗寒生理指标的隶属函数综合评价,结果表明,26个新品系的抗寒性分为4个等级,其中抗寒能力强的为崇枇10号、12号、20号、21号、26号,抗寒能力弱的为崇枇2号、3号、5号、18号、23号,其余品系表现为较强或中等抗寒性,整体表现出较高的抗寒性。在低温胁迫下,崇枇3号、18号、23号的相对电导率变幅最大,崇枇12号、20号、26号变幅最小;崇枇12号与崇枇20号丙二醛含量均值最低(13.00μmol/g.10^-3、13.53μmol/g.10^-3),崇枇21号脯氨酸含量最高（62.21μg/g.dw）、崇枇18号最低（36.03μg/g.dw）,崇枇26号可溶性糖含量最高（89.23 mg/g）,崇枇2号、3号最低（46.54 mg/g、53.89 mg/g）,崇枇26号SOD、POD、CAT活性最高,崇枇18号、23号最低。4.光合性能研究显示,各供试新品系的Pn、Gs、Ci、Tr、WUE、CE差异极显著（p<0.01）,变异系数为Gs>CE>Pn>WUE>Tr>Ci。崇枇26号的净光合速率最高,Pn=13.20μmol/m²·s,比对照品种福鼎大白高42.70%。采用因子判别归类法和聚类分析法评价品系光能利用率和水分利用效率,光能利用率最高的品系为崇枇8号、22号、25号、26号,具有选育为高产茶树品种的潜力;水分利用率最高的为崇枇4号、15号、18号,具有选育为抗旱茶树品种的潜力。5.代谢组学分析显示,-10℃下处理2h、4h、8h、16h、24h,抗寒性最强的崇枇26号品系与抗寒性最弱的崇枇23号品系代谢谱发生显著变化,差异显著代谢物总数953个,其中上调表达代谢物372个,下调表达代谢物581个,差异代谢物主要有氨基酸类相关物质、糖及糖类衍生物,主要富集在半乳糖代谢、淀粉和蔗糖代谢、戊糖和葡萄糖醛酸互作、糖酵解/糖异生、精氨酸和鸟氨酸代谢、苯丙氨酸、酪氨酸和色氨酸生物合成、β-丙氨酸代谢等糖代谢和氨基酸合成代谢途径。低温胁迫下,氨基酸类物质中脯氨酸和蛋氨酸类相关物质,以及棉籽糖、蔗糖、水苏糖、L-阿拉伯糖醇上调倍数较高,可能是崇州枇杷茶响应低温胁迫的主要物质。6.转录组学分析显示,-10℃下处理2h、4h、8h、16h、24h,抗寒性强的品系崇枇26号与抗寒性弱的品系崇枇23号基因表达发生显著变化,差异表达基因总数13369个,其中上调表达基因7211个,下调表达基因6158个,主要富集在植物病原互作、RNA运转、乙醛酸和二羧酸代谢、丙氨酸、天冬氨酸和谷氨酸代谢、柠檬酸循环、氮代谢。低温胁迫下,参与糖类代谢基因RS2、BAM、AMY、α-半乳糖苷酶、β-半乳糖苷酶、MFS、ERD6、SWEET、HXK的基因,Ca²⁺信号通路基因CDPK、CBL、CIPK、Ca M、CNGC、TPC,转录因子WRKY、MYB、b ZIP、b HLH、AP2/ERRBP、NAC表达差异显著,可能是崇州枇杷茶响应低温胁迫的主要基因。7.代谢组与转录组联合分析解析崇州枇杷茶低温胁迫响应机制,结果表明,-10℃下处理2h、4h、8h、16h、24h,抗寒性强的品系崇枇26号与抗寒性弱的品系崇枇23号中棉籽糖合成酶相关基因Camellia＿new Gene＿6317、Cha Un25108.1、半乳糖苷酶合成基因Cha03g009050、Cha Un5396.6、编码鸟氨酸转氨酶的基因Camellia＿sinensis＿new Gene＿28159、吡咯啉-5-羧酸还原酶Camellia＿sinensis＿new Gene＿22006、Cha15g004060基因表达变化趋势与相关代谢物含量变化趋势基本一致。综上所述,26个崇州枇杷茶新品系处于野生型茶树向栽培型茶树过渡阶段,其表型及遗传多样性水平较高,且整体表现出生长势强、早生、高茶多酚和儿茶素、抗寒性强的特点,其中崇枇11号、12号、23号、26号总体性状较优,具有选育高产、特色（高茶多酚或高儿茶素、特早生）和耐寒性强的茶树品种的潜力;崇州枇杷茶在低温胁迫下可能是通过上调Camellia＿new Gene＿6317、Camellia＿sinensis＿new Gene＿22006、Cha Un25108.1、Cha03g009050、Cha Un5396.6、Cha15g004060等基因表达,促进棉籽糖、水苏糖和脯氨酸类在体内积累,从而提高抵御低温的能力。更多还原

【Abstract】 Sichuan is not only one of the birthplaces of tea plants,but also the second largest province of tea planting area in China.Tea germplasm resources are very rich,which has laid a solid foundation for the cultivation of high-quality new varieties of tea.Chongzhou loquat tea（CZLT）,also known as Chongqing loquat tea,is a local tea cultivars formed through long-term natural evolution and semi-artificial domestication,has the characteristics of strong branching capacity,strong adaptability,strong cold tolerance and large leaves similar to loquat leaves.However,due to natural disasters,man-made logging,overexploitation and other factors,the number of wild CZLT has decreased sharply.In order to protect,develop and utilize the resources of CZLT,the research team has selected 26 new CZLT strains from its excellent individual plants based on systematic breeding method for decades.In order to further develop this excellent local tea resource,in this paper,the above-mentioned new strains were multiply evaluated through whose morphological characteristics,physiological characteristics,photosynthetic performance,cold resistance ability,high throughput transcriptome sequencing and metabonomics were used to evaluate the molecular regulation and metabolic response mechanism of cold resistance to provide a scientific theoretical basis for the cultivation of new varieties of Chongzhou loquat tea with excellent cold resistance and quality.The results are as follows:1.The 26 new CZLT strains mainly showed the characteristics of evolution from the wild type to the cultivated type,such as elliptical leaf shape and gradually pointed leaf tip,but there were still some wild-type tea characters,such as big leaves and big flowers,sparse and blunt leaf teeth,raised leaf surface and no hair in ovary.The mean variation coefficient of the morphological traits of the new strains was 34.17%,and its diversity index was 0.70.The genetic diversity level based on SSR marker was PIC=0.636,indicating that the new strains of CZLT had a high level of phenotype and genetic diversity,and its morphological and phenotypic stability was Bud>leaf>tree>flower.The one hundred bud weight of one bud and three leaves,leaf length,leaf width and the other yield-related factors had a higher genetic differentiation level,the new strains were potential tea resource for breeding for high-yield tea varieties.CZLT 24,26,23,20 and 16 showed strong growth potential,and one hundred buds weight of one bud and three leaves was relatively high,which had the potential to breed high-yield tea varieties.The buds and leaves of CZLT 3,5,11,and 20 were purple,which had the potential to breed purple tea varieties and high-anthocyanin tea varieties.CZLT 3,5,11,17 and 20 belonged to special tea plant germplasm resources.The germinating dates of CZLT 13 and 26 were 12 and 10 days earlier than CK,respectively,which indicated that such strains belonged to the early-germination tea strains.2.The polyphenols（TP）content of new shoots of CZLT strains ranged from 16.75%to28.28%,the content of TP in CZLT 3,11 and 23 in spring,summer and autumn was all>25%,indicating that which were specific tea strains with high TP.The amino acid content of new shoots of CZLT strains ranged from 1.74%to 5.69%,of which,the total amino acid content of new shoots of CZLT 25 and 26 was 5.69%and 5.18%,respectively,indicating they were high amino acid tea shoots.The caffeine content of new shoots was 3.24%-5.29%,and the content of CZLT 2 and 10 was 5.03%and 5.29%,respectively,which met the standard of high caffeine tea germplasm resources.The average values of catechins in CZLT11,12,22 and 23 at spring,summer and autumn were 23.04%,21.69%,23.48%and23.90%,respectively,they were belonging to the tea strains with high catechins.Additionally,the average values of EGCG content in CZLT 11,12,22,and 23 at different seasons was16.25%,15.31%,15.92%,and 15.95%,respectively,which indicated that such new strains belonged to the high EGCG tea germplasm resources.3.The results of photosynthetic performance research showed that Pn,Gs,Ci,Tr,WUE and CE of all the new CZLT strains were significantly different（P<0.01）,and the C.V.was Gs>CE>Pn>WUE>Tr>Ci.CZLT 14 had the highest net photosynthetic rate（Pn=11.57μmol/m²·s）.Twenty-six new CZLT strains were clustered into three different Pn groups,and the differences reached a significant level.The three strains with the highest light energy utilization were CZLT 8,22,25,and 26,which had the potential to be selected as high-yield tea varieties,the highest water utilization rate was CZTL 4,18,and 15,which had the potential to be a drought resistant tea variety.4.The cold resistance of the new CZLT strains was determined in the field and the laboratory.The results showed that the cold resistance of the 26 strains could be divided into4 grades,of which CZLT 10,12,20,21,and 26 had strong cold resistance,while CZLT 2,3,5,18,and 23 had weak cold resistance.The other strains showed relatively strong or medium cold resistance.In the low temperature treatment,the relative conductivity of CZLT 3,18,and 23 changed the most,and 12,20,and 26 changed the least.Under the conditions of natural growth and low temperature treatment,the average content of malondialdealdehyde of CZLT12 and 20 were the lowest(13.00μmol/g10^-3 and 13.53μmol/g10^-3),the highest content of proline existed in CZLT 21（62.21μg/g W）and the lowest content of which was discovered in CZLT 18（36.03g/g W）.The content of soluble sugars was the highest in CZLT26（89.23 mg/g）,and the lowest in CZLT 2 and 3（46.54 mg/g and 53.89 mg/g）.The activities of SOD,POD and CAT were the highest in CZLT26,and the lowest in CZLT 18 and 23.After comprehensive evaluation by the membership function method,CZLT12,20,21,and26 could be used as cold-tolerant tea strains.5.Metabonomics analysis showed that after treatment at-10℃for 2h,4h,8h,16h,and24h,the metabolic profile of CZLT 26（the strong cold resistance strain）and 23（the weak cold resistance strain）changed significantly,and the total number of significantly different metabolites is 953,of which 372 were up-regulated and 581 were down-regulated.Among them,8 metabolites such as leucine methionine,3-glucosyl-patchoulin,7,2’-dimethoxy-4’,5’-methylenedioxy isoflavone,1-salicylic acid glucoside acid,2,5-dihydroxybenzaldehyde,epicatechin,shikimic acid and thymine were significantly different at 5 time points.Different metabolites were concentrated in amino acids,sugars and sugar derivatives.The pathways of different metabolites enrichment were mainly concentrated in galactose metabolism,starch and sucrose metabolism,interaction between pentose and glucuronic acid,glycolysis/gluconeogenesis,arginine and ornithine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,β-alanine metabolism and so on.6.Transcriptomics analysis showed that after treatment at-10℃for 2h,4h,8h,16h and24h the gene expression of CZLT 26 and 23 changed significantly,and a total of 13369 genes differentially expressed,of which 7211 genes up-regulated and 6158 genes down-regulated,among them,1871 genes were significantly different at 5 time points.Genes with significant differences were mainly enriched in plant pathogen interaction,RNA translocation,glyoxylic acid and dicarboxylic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,citric acid cycle,nitrogen metabolism and other pathways,and involved in cell physiological process,metabolic process,membrane,membrane part,binding,catalytic activity and other pathways.Under low temperature stress,the genes involved in carbohydrate metabolism including RS2,BAM,AMY,α-galactosidase,β-galactosidase,MFS,ERD6,SWEET,HXK,and Ca²⁺signaling pathway genes CDPK,CBL,CIPK,Ca M,CNGC,TPC,and transcription factors WRKY,MYB,b ZIP,b HLH,AP2/ERRBP,NAC had significant differences in expression,which may play an important role in the response of CZLT to low temperature stress.In summary,based on the results of morphological characteristics,physiological characteristics,photosynthetic performance,cold resistance characteristics,transcriptomics and metabolomics research of the new CZLT,CZLT 3,11,12,22,23 and 26 were the best among the 26 Chongzhou loquat tea lines.Chongpi No.3 can be further bred as purple,early-growing and high tea polyphenol tea variety,chongpi No.11 can be further bred as purple,high tea polyphenol,high catechin,high EGCG and strong cold resistance tea variety,chongpi No.12 can be further bred as high catechin,high EGCG and strong cold resistance tea variety,chongpi No.22 can be further bred as high catechin,high EGCG and high light efficiency tea variety Chongpi 23 can be further selected as Zaosheng tea variety with high tea polyphenols,catechins and EGCG,while chongpi 26 can be further selected as Zaosheng tea variety with high amino acids,high light efficiency and strong cold resistance.更多还原