【作者】 王慧；

【导师】 王玉成； 李丹丹； 赵福强；

【作者基本信息】 沈阳农业大学 ， 林业硕士（专业学位）， 2024， 硕士

【摘要】 林木在生长发育过程中会受到多种非生物胁迫影响,其中干旱是严重影响我国农林业的产出和发展的因素之一。分析林木的抗旱分子机制和提高林木的抗旱性对林业的发展具有重要的意义。C₂H₂型锌指蛋白是植物物种中最为广泛的转录因子之一,其在植物应对非生物胁迫过程中扮演着极其重要的角色。本文对山新杨（Populus davidiana×Populus bolleana）PdbZAT10基因在干旱胁迫下进行了初步研究分析,结果如下:（1）从山新杨中克隆了PdbZAT10基因,其片段长度为747 bp,共编码248个氨基酸。根据生物信息学分析,PdbZAT10蛋白含有典型的C₂H₂锌指蛋白结构域,是C₂H₂型锌指转录因子C1-2i亚类的关键成员,该亚类包含两个不同的C₂H₂型锌指和一个EAR结构域;进化树分析结果表明PdbZAT10与拟南芥At ZAT10亲缘关系较近;荧光定量PCR（q RT-PCR）结果表明在干旱胁迫下,PdbZAT10基因在叶中的表达量最高,在胁迫1 h时达到峰值。说明PdbZAT10基因能够被干旱胁迫诱导。（2）利用基因枪法瞬时转化洋葱表皮细胞进行亚细胞定位实验,发现PdbZAT10基因定位在细胞核。构建了pGBKT7-PdbZAT10酵母表达载体,酵母转录激活活性实验结果表明PdbZAT10蛋白无转录激活活性。（3）分别构建pBI121-PdbZAT10-GFP植物表达载体以及CRISPR/Cas9-PdbZAT10基因编辑载体,获得了pBI121-PdbZAT10-GFP（PdbZAT10-OE）过表达转基因植株。与WT相比,PdbZAT10-OE株系较矮,叶面积较大且根茎比较大。对WT和PdbZAT10-OE株系土培苗进行干旱胁迫,通过电导率、MDA、H₂O₂、脯氨酸含量和三种化学染剂（DAB、NBT和Evens blue）分析得出在干旱胁迫下,PdbZAT10-OE株系的细胞受损程度明显低于WT,PdbZAT10基因可以提高山新杨的抗旱性。（4）对正常生长和干旱胁迫7 d的WT和PdbZAT10-OE株系的叶片进行转录组测序。正常生长条件下共获得1337个差异基因,其中有399个基因上调表达,938个基因下调表达;干旱胁迫后共有6518个差异基因,其中有4120个基因上调表达,2398个基因下调表达。对差异基因进行GO分类和KEGG富集分析,发现其与代谢途径和应激反应相关,如细胞壁生物合成、叶绿素代谢过程、响应赤霉素等。（5）构建pGADT7-Rec2-PdbZAT10酵母表达载体,与随机酵母元件文库进行酵母单杂交（Y1H）,结果发现PdbZAT10蛋白能够与三个未知功能的元件结合,分别为PdbZAT10-2motif（TATCGGG）、PdbZAT10-4motif（CGCCGGG）和PdbZAT10-7motif（CGATCCT）,进一步确定其核心序列分别为ATCGG、GCCG和GATC。本研究为山新杨抗旱分子机制提供了新的思路,对今后杨树育种改良具有重要意义。更多还原

【Abstract】 During the growth and development of trees,they are affected by various abiotic stresses,among which drought is one of the serious factors affecting the output and development of China’s agriculture and forestry industry.Analyzing the molecular mechanisms of drought resistance in trees and improving their drought resistance is of great significance for the development of forestry.C₂H₂ type zinc finger protein is one of the most widely studied transcription factors in plant species,playing an extremely important role in plant response to abiotic stress.This article conducted a preliminary study and analysis on the PdbZAT10 gene of Populus davidiana×Populus boleana（Shanxin poplar）under drought stress,and the results are as follows:（1）The PdbZAT10 gene was cloned from P.davidiana×P.bolleana,with a fragment length of 747 bp and encoding a total of 248 amino acids.According to bioinformatics analysis,the PdbZAT10 protein contains a typical C₂H₂ zinc finger protein domain,which is a key member of the C₂H₂ type zinc finger transcription factor C1-2i subclass.This subclass contains two different C₂H₂ type zinc fingers and an EAR domain,indicating that the PdbZAT10 gene is a transcription inhibitor.The evolutionary tree analysis shows that the PdbZAT10 is closely related to At ZAT10.This study subjected the roots,stems,and leaves of P.davidiana×P.bolleana to 6%PEG6000 stress at different time points（0 h,1 h,3 h,6 h,12 h,24 h,48 h,and 72 h）,and analyzed their expression patterns.It was found that under drought stress,the expression level of the PdbZAT10 gene was highest in the leaves,reaching its peak at1 h of stress.This indicates that the PdbZAT10 gene can be induced by drought stress.（2）This study used the gene gun method to instantaneously transform onion epidermal cells for subcellular localization experiments,and found that the PdbZAT10gene was located in the nucleus.We constructed pGBKT7-PdbZAT10 yeast expression vector and verified the non transcriptional activation activity of the PdbZAT10 protein through yeast transcriptional activation experiments.（3）This study constructed plant expression vectors pBI121-PdbZAT10-GFP and CRISPR/Cas9-PdbZAT10 gene editing vectors,and obtained transgenic plants overexpressing pBI121-PdbZAT10-GFP.After natural drought stress on soil cultured seedlings of WT and PdbZAT10-OE strains,it was found that compared with WT,the PdbZAT10-OE strain was shorter,had larger leaf area,lower leaf water loss rate,and larger root and stem ratio.Through analysis of conductivity,MDA,H₂O₂,proline content,and three chemical dyes（DAB,NBT,and Evans blue）,it was found that under drought stress,the degree of cell damage in the PdbZAT10-OE strain was significantly lower than that in the WT strain.It is preliminarily concluded that the PdbZAT10 gene can improve the drought resistance of Shanxin poplar.（4）Transcriptome sequencing was performed on the leaves of WT and PdbZAT10-OE strains under normal growth and natural drought stress for 7 days.In the leaves,a total of 1337 differentially expressed genes were obtained under normal conditions,of which 399 genes were upregulated and 938 genes were downregulated;After stress,there were a total of 6518 differentially expressed genes,of which 4120 genes were upregulated and 2398 genes were downregulated.GO classification and KEGG enrichment analysis were performed on differentially expressed genes,and it was found that they are related to metabolic pathways and stress responses,such as cell wall biosynthesis,chlorophyll metabolism and response to gibberellins.（5）Construct a pGADT7-Rec2-PdbZAT10 yeast expression vector and perform yeast single hybridization（Y1H）with a random yeast element library.The results showed that the PdbZAT10 protein can bind to three unknown functional elements,namely PdbZAT10-2motif（TATCGGG）,PdbZAT10-4motif（CGCCGG）,and PdbZAT10-7motif（CGATCCT）.Further confirm that its core sequences are ATCGG,GCCG and GATC.This study provides new insights into the molecular mechanisms of drought resistance in Shanxin poplar and is of great significance for future poplar breeding and improvement.更多还原