【作者】 陈蓉；

【导师】 黄小贞；

【作者基本信息】 贵州大学 ， 生物学， 2022， 硕士

【摘要】 茶树(Camellia sinensis L.)是世界重要的经济作物之一,优良茶树品种的快速繁殖对于现代化茶产业的发展至关重要。茶树扩繁主要通过短穗节段进行扦插,不定根(Adventitious root,AR)的形成和发育是插穗存活的关键。目前,有关茶树动态变化且连续发展的不定根形成分子调控机制仍然不清楚。为了进一步解析茶树不定根发育调控网络,本研究以生根能力强的‘苔选0310’(C.sinensis.cv.Taixuan,‘TX’)和生根能力弱的‘黔湄601’(C.sinensis.cv.Qianmei 601,‘QM601’)为研究材料,利用转录组测序技术分析两个品种不定根不同发育阶段基因的动态表达变化。并从差异表达基因中筛选克隆了一个可能在不定根形成过程中起重要作用的GH3(GRETCHEN HAGEN3)基因,命名为Cs GH3.3。主要研究结果如下:1、本研究发现8种不同的茶树品种,其不定根形成能力存在显著差异。统计数据显示,TX成活率为97%,生根率为96%,鲜重为3.43±0.37g,干重为1.38±0.23g,显著优于其他7个品种。QM601成活率为85%,生根率为79%,鲜重为2.14±0.44g,干重为0.89±0.22g,与TX存在极显著差异。该结果表明TX和QM601可作为研究不定根形成的理想品种。2、本研究对TX和QM601三个根系发育时期(S1,S2和S3)的组织进行了转录组测序分析,总共有9个比较组。其中,TX-S1与TX-S2、TX-S1与TX-S3、TX-S2与TX-S3这三个比较组中筛选到的差异基因分别和QM-S1与QM-S2、QM-S1与QM-S3、QM-S2与QM-S3这三个比较组的差异基因表现为相似的基因富集模式。该结果表明,不同茶树品种,可能存在着相似的不定根形成机制。3、对TX-S1与TX-S2和TX-S1与TX-S3两个比较组筛选到的2907个共同差异基因进行GO分析发现,大部分基因富集于细胞过程、代谢过程和刺激响应等过程。KEGG富集主要映射到苯丙烷生物合成、植物激素信号转导、植物MAPK信号通路和植物病原体相互作用四条途径。该结果表明,这四个途径在茶树不定根形成过程中起重要作用。研究中筛选获得的大量差异表达基因,为可能参与茶树插穗内部发育信号诱导AR形成的基因网络提供了一个全局视图。4、分析TX-S1与TX-S3比较组的差异表达基因发现,多个生长素合成代谢相关基因的表达在不同的生根阶段存在显著差异。其中,Cs GH3.3基因在样本S2和S3阶段中高表达,但在阶段S1中表达量非常低。进一步从TX中克隆得到了该基因的编码区,测序结果显示,Cs GH3.3基因开放阅读框包含1794个核苷酸,编码598个氨基酸。基序分析和BLAST比对结果表明,Cs GH3.3包含一个保守的“GH3 auxin-responsive promoter”结构域。与参考基因组相比,克隆获得的Cs GH3.3编码区序列具有多个SNP,但是这些SNP突变均没有改变氨基酸序列,这表明茶树中Cs GH3.3的功能可能较为保守。利用DNAMAN进行序列比对,结果显示Cs GH3.3与拟南芥At GH3.1(AT2G14960)的相似度为81.27%,与水稻的Os GH3.8(LOC＿Os07g40290.1)相似性为71.95%。5、荧光定量PCR分析Cs GH3.3在正常条件下不同组织和器官中的表达模式发现,其在茶树的根部组织中表达量最高。外源激素IBA处理1小时后,Cs GH3.3在根部迅速上调。该结果表明,Cs GH3.3能够响应生长素,有可能参与生长素依赖的不定根形成过程。另外,亚细胞定位结果表明,Cs GH3.3蛋白在烟草表皮细胞中定位于叶绿体。6、本研究进一步通过农杆菌介导的水稻愈伤转化方法获得30株Cs GH3.3基因过表达的转基因水稻。转化双子叶三星烟草,但没有获得转基因植株。对过表达Cs GH3.3水稻株高、分蘖数等农艺性状进行了统计,结果发现,与野生型中花11相比,转基因水稻植株地上部和地下部均变小,具有矮小和簇状的表型,但分蘖数增加,发育不良表型的程度与Cs GH3.3过表达水平密切相关;转基因烟草生根困难。除此之外,还发现转基因水稻不育。进一步的细胞学观察显示,Cs GH3.3过表达植株的根尖细胞大小和细胞数量与野生型相比显著减少。这些研究结果初步验证了Cs GH3.3作为一个有功能的GH3,参与了植物根系的形成发育。更多还原

【Abstract】 Tea(Camellia sinensis L.)is a commercial cash crop used to produce one of the most popular and economically valuable beverages worldwide.The most efficient and economical way to produce large numbers of homogeneous clonal tea plants are to through nodal cuttings The formation of adventitious roots(ARs)is essential for successful propagation and long-term survival of cuttings.ARs formation is thus essential for the successful propagation and long-term survival of tea plants.Numerous important players involved in AR development have been identified in the model plants Arabidopsis and rice.However,the molecular mechanisms behind the successive,irreversible steps of the rooting process of tea cuttings are still unclear.The present study identified two tea cultivars with different rooting behavior:Camellia sinensis cv.Taixuan 0310(TX0310),with strong and rapid rooting;and C.sinensis cv.Qianmei 601(QM601),with poor and slow rooting.To elucidate the molecular events underlying AR formation in tea cuttings,transcriptomic analysis was performed during AR formation in tea cuttings,from root callus formation to root initiation to root elongation.More DEGs were observed during phase transitions than during the various growth stages.The differences in gene expression patterns were examined in primary calli and re-differentiated roots in two tea cultivars with different rooting abilities.Among the DEGs,one GRETCHEN HAGEN3(GH3)genes,Cs GH3.3,was isolated and characterized.The main findings are as follows:1.This study found that there were significant differences in the formation of adventitious roots among 8 different tea varieties.Statistics showed that the survival rate of TX was 97%,the rooting rate was 96%,the fresh weight was 3.43±0.37 g,and the dry weight was 1.38±0.23 g,which was significantly better than the other 7varieties.The survival rate of QM601 was 85%,the rooting rate was 79%,the fresh weight was 2.14±0.44 g,and the dry weight was 0.89±0.22 g,which was significantly different from TX.The results indicated that TX and QM601 could be used as ideal cultivars for studying adventitious root formation.2.In this study,we performed transcriptome sequencing analysis of tissues from three root developmental stages(S1,S2,and S3)of TX and QM601,with a total of 9comparison groups.Among them,the differential genes screened in the three comparison groups of TX-S1 and TX-S2,TX-S1 and TX-S3,and TX-S2 and TX-S3 were the same as those of QM-S1 and QM-S2,and QM-S1 and TX-S3,respectively.The differential genes of the three comparison groups QM-S3,QM-S2 and QM-S3 showed similar gene enrichment patterns.The results indicated that different tea varieties may have similar adventitious root formation mechanisms.3.GO analysis of the 2907 common differential genes screened from the two comparison groups of TX-S1 and TX-S2 and TX-S1 and TX-S3 found that most of the genes were enriched in cellular processes,metabolic processes and stimuli response.KEGG analysis enrichment mainly mapped to four pathways,including phenylpropane biosynthesis,plant hormone signaling,plant MAPK signaling,and plant pathogen interaction.This result suggests that these four pathways play important roles in the formation of adventitious roots of tea plants.The large number of differentially expressed genes screened in this study provide a global view of the gene network that may be involved in the formation of AR induced by internal developmental signals in tea plant cuttings.4.Analysis of differentially expressed genes between TX-S1 and TX-S3 comparison groups found that there were significant differences in the expression of several auxin synthesis and metabolism-related genes at different rooting stages.Among them,the Cs GH3.3 gene was highly expressed in the samples S2 and S3 stages,but the expression level was very low in stage S1.The coding region of the gene was further cloned from the tea variety TX.The sequencing results showed that the open reading frame of the Cs GH3.3 gene contained 1794 nucleotides,encoding598 amino acids.Motif analysis and BLAST alignment showed that Cs GH3.3contained a conserved domain "GH3 auxin-responsive promoter".Compared with the reference genome,the cloned Cs GH3.3 coding sequence had multiple SNPs,but none of these SNP mutations changed the amino acid sequence,which indicated that the function of Cs GH3.3 in Camellia sinensis may be conserved.The sequences of related members of the GH3 family by DNAMAN,and the results showed that Cs GH3.3 was 81.27% similar to Arabidopsis At GH3.1(AT2G14960),and Os GH3.8(LOC＿Os07g40290.1)of rice,The similarity is 71.95%.5.q RT-PCR analysis of the expression pattern of Cs GH3.3 in different tissues and organs under normal conditions found that its highest expression was in the root tissue of tea plant.After 1 hour of exogenous hormone IBA treatment,Cs GH3.3 was rapidly up-regulated in roots.The results indicate that Cs GH3.3 can respond to auxin and may be involved in auxin-dependent adventitious root formation.In addition,the subcellular localization results showed that the Cs GH3.3 protein was localized to the chloroplast in tobacco epidermal cells.6.In this study,30 strains of transgenic rice with overexpression of Cs GH3.3gene were obtained by Agrobacterium-mediated rice callus transformation.Dicotyledonous three-star tobacco was transformed,but no transgenic plants were obtained.The agronomic traits such as plant height and tiller number of rice overexpressing Cs GH3.3 were counted,and it was found that compared with wild-type ZH 11,the above-ground and underground parts of the mutant rice plants were smaller,with dwarf and clustered ones.phenotype,but the number of tillers increased,and the degree of stunted phenotype was closely related to the overexpression level of Cs GH3.3;transgenic tobacco had difficulty in rooting.In addition,mutant rice was also found to be sterile.Further cytological observation showed that the root tip cell size and cell number of Cs GH3.3 overexpressing plants were significantly reduced compared with wild type.These findings preliminarily verify that Cs GH3.3,as a functional GH3,is involved in the formation and development of plant roots.更多还原