【作者】 杨旭；

【导师】 王丰青；

【作者基本信息】 河南农业大学 ， 作物学， 2023， 硕士

【摘要】 为了研究地黄苯乙醇苷类成分生物合成的调控机制,对地黄全长转录组进行了测序,鉴定出参与苯乙醇苷类成分合成的催化酶基因的序列,分析了催化酶基因的表达特性及其与毛蕊花糖苷合成的相关性。应用水杨酸(SA)及低温处理研究非生物诱导对地黄苯乙醇苷含量的影响和相关基因转录特性,测定了水杨酸和低温处理后地黄的生理生化指标变化、药效成分含量变化,并应用转录组测序技术分析了相关基因的表达模式。利用过量表达和基因编辑技术初步分析了RgWRKY35在地黄苯乙醇苷合成中的调控功能。主要研究内容如下:1.以地黄的叶、茎和块根为材料,利用Pacific Biosciences RS II平台进行全长转录组测序。共获得非冗余的转录本27 773条,平均长度2 380 bp,预测出27 236个CDS。利用BLAST等软件在NR、NT、GO、COG、KEGG、Swissprot和Interpro等数据库共预测到27399个注释的基因。NR注释表明,与地黄转录本匹配数量最多的是芝麻,有81.44%。推测了参与异毛蕊花糖苷、松果菊苷、肉苁蓉苷A、肉苁蓉苷F、2-乙酰毛蕊花糖苷和Leonoside F生物合成的催化酶,并鉴定出143个参与苯乙醇苷类成分生物合成的转录本。19个催化酶基因在地黄12个组织中与毛蕊花糖苷的含量呈正相关,其中多数基因在叶和花中具有较高的表达量。2.以地黄栽培品种温85-5为材料,用SA喷施生长180 d的地黄植株,在处理后的0h、1 h、3 h和6 h收集地黄叶片和块根,测定毛蕊花糖苷及总苯乙醇苷的含量,并对SA处理后不同时间的块根进行转录组测序分析。结果显示,SA处理后地黄叶片中的毛蕊花糖苷提高了11.2～19.3%,块根中毛蕊花糖苷的含量提高了0.9～1.4倍。叶片中的总苯乙醇苷含量在SA处理后6 h比对照提高28.5%,块根中总苯乙醇苷含量在处理后分别比对照提高了0.65～0.82倍。转录组分析表明,苯乙醇苷生物合成通路在差异表达的基因中得到显著富集,ALDH基因、UGT基因和PPO等少数毛蕊花糖苷合成途径的催化酶基因在SA处理后的地黄块根中上调表达。筛选并鉴定出多个在SA处理后差异表达的AP2-EREBP、WRKY和MYB转录因子基因。3.利用人工智能气候箱对地黄品种QH-1和BZY进行不同温度处理,在5°C和15°C处理下地黄叶片中的抗氧化酶活性呈先升高后下降的趋势,脯氨酸含量先上升后降低,可溶性糖含量整体上呈上升趋势。测定了不同温度处理后地黄有效成分含量的变化,结果表明,适度低温促进总苯乙醇苷和毛蕊花糖苷含量的积累。对低温胁迫的地黄叶片进行转录组测序,结果显示低温胁迫后差异表达的基因主要富集在淀粉与蔗糖代谢、单萜生物合成、吲哚生物碱生物合成、类胡萝卜素生物合成、类黄酮生物合成等代谢通路。分析了低温处理后富集到苯乙醇苷生物合成通路的催化酶基因表达特性,筛选了响应低温参与苯乙醇苷和梓醇合成的候选基因,分析了低温处理后类黄酮生物合成途径差异表达的基因。4.利用聚合酶链式反应技术(PCR)克隆了地黄RgWRKY35基因,其编码区序列(CDS)长度894 bp,编码297个氨基酸。多序列联配表明RgWRKY35具有典型的WRKY结构域。亚细胞定位分析发现RgWRKY35蛋白分布在细胞核中,属于核蛋白。利用农杆菌介导法将RgWRKY35基因的过量表达载体和基因编辑载体导入地黄基因组,获得了过量表达和基因编辑的转化地黄植株。过量表达RgWRKY35基因的地黄株系毛蕊花糖苷含量显著升高,基因编辑RgWRKY35的地黄株系毛蕊花糖苷含量显著降低,表明RgWRKY35基因是地黄苯乙醇苷类成分毛蕊花糖苷的正向调控因子。更多还原

【Abstract】 To study the regulatory mechanism of phenylethanoid glycoside biosynthesis in Rehmannia glutinosa,the full-length transcriptome of R.glutinosa was sequenced,and the sequence of the catalytic enzyme genes involved in phenylethanoid glycoside biosynthesis was identified.The expression characteristics of the catalytic enzyme genes and their correlation with acteoside biosynthesis were analyzed.Salicylic acid(SA)and low-temperature treatment were used to study the effects of abiotic induction on the content of phenylethanoid glycosides in R.glutinosa and the transcription characteristics of related genes.The changes in physiological and biochemical indicators and the content of pharmacological components of R.glutinosa after SA and low-temperature treatment were measured,and the expression patterns of related genes were analyzed using transcriptome sequencing technology.The regulatory function of RgWRKY35 in the synthesis of phenylethanoid glycoside of R.glutinosa was preliminarily analyzed using overexpression and gene editing techniques.The main research contents are as follows:1.The leaves,stems and tuberous roots of R.glutinosa were used for transcriptome sequencing by Pacific Biosciences RS II platform.A total of 27 773 transcripts were generated with an average length of 2 380 bp,and 27 236 coding sequences(CDS)were predicted.Using the BLAST software,non-redundant transcript sequences were annotated with NR,NT,GO,COG,KEGG,Swiss Prot and Interpro databases and a total of 27 399 annotated genes were obtained.Among them,the number of genes related to Sesamum indicum in the NR database was the highest(81.44%).Enzymes likely involved in the biosynthesis of isoacteoside,echinacoside,cistanosides A,cistanosides F,2’-acetylacteoside and leonoside F were identified,and 143 genes were identified in R.glutinosa full-length transcriptome.The expression levels of 19 genes correlated with acteoside content in twelve tissues of R.glutinosa,and most showed higher expression levels in leaf tissues and floral organs.2.The cultivar Wen 85-5 of R.glutinosa was used as material,and SA was sprayed on R.glutinosa plants growth to 180 days,at 0 h、1 h、3 h and 6 h after treatment,the leaves and tuberous roots of R.glutinosa were collected to determine the content of acteoside and total phenylethanoid glycoside,and the transcriptome sequencing analysis of tuberous roots at different times after SA treatment was performed.The results showed that after SA treatment,the content of acteoside in leaves of R.glutinosa increased by 11.2 to 19.3 %,and the content of acteoside in the tuberous root increased by 0.9 to 1.4 times.The content of total phenylethanoid glycoside in leaves increased by 28.5 % compared to the control 6 hours after SA treatment,and the content of total phenylethanoid glycoside in the tuberous root increased by 0.65 to 0.82 times compared with the control.Transcriptome analysis showed that the phenylethanoid glycoside biosynthesis pathway was significantly enriched among the differentially expressed genes,and a few catalytical enzyme genes of the acteoside synthesis pathway,such as ALDH,UGT and PPO,were up-regulated in the tuberous roots of R.glutinosa treated with SA.A lot of AP2-EREBP,WRKY and MYB transcription factor genes that were differentially expressed after SA treatment were screened and identified.3.Using an artificial intelligence climate box,R.glutinosa varieties QH-1 and BZY were subjected to different temperature treatments.Under the treatment of 5 °C and 15 °C,the antioxidant enzyme activity in R.glutinosa leaves first increased and then decreased,the proline content first increased and then decreased,and the soluble sugar content overall showed an upward trend.The changes in the content of active components of R.glutinosa after different temperature treatments were measured.The results showed that moderately low temperatures promoted the accumulation of total phenylethanoid glycoside and acteoside contents.Transcriptome sequencing of R.glutinosa leaves under low-temperature stress showed that the differentially expressed genes after low-temperature stress were mainly concentrated in metabolic pathways such as starch and sucrose metabolism,monoterpene biosynthesis,indole alkaloid biosynthesis,carotenoid biosynthesis,and flavonoid biosynthesis.The expression characteristics of catalytic enzymes enriched in phenylethanoid glycoside biosynthesis pathway after low-temperature treatment were analyzed,the candidate genes involved in phenylethanoid glycoside and catalpol biosynthesis in response to low-temperature treatment were screened,and differentially expressed genes in flavonoid biosynthesis pathway after low-temperature treatment were analyzed.4.The RgWRKY35 gene from R.glutinosa was cloned using polymerase chain reactions(PCR).The coding region sequence of the RgWRKY35 gene was 894 bp in length and encoded 297 amino acids.Multiple sequence alignment indicated that RgWRKY35 has a typical WRKY domain.Subcellular localization analysis revealed that the RgWRKY35 protein is distributed in the nucleus and belongs to the nuclear protein.The overexpression vector and gene editing vector of the RgWRKY35 gene were introduced into the genome of R.glutinosa by the Agrobacterium tumefaciens mediated method,and the overexpression and gene editing transformed R.glutinosa plants were obtained.The content of acteoside in R.glutinosa strains overexpressing the RgWRKY35 gene significantly increased,while the content of acteoside in R.glutinosa editing the RgWRKY35 gene significantly decreased,indicating that the RgWRKY35 gene is a positive regulator of acteoside,a phenylethanoid glycoside component of R.glutinosa.更多还原