【作者】 张琪；

【导师】 黄镇； 徐爱遐；

【作者基本信息】 西北农林科技大学 ， 作物学， 2022， 硕士

【摘要】 甘蓝型油菜（Brassica napus L.）作为我国油菜最主要的栽培类型,其产量和品质对维持粮食市场的安全稳定性具有重要意义。随着世界人口的增加,人均可利用耕地面积日益减少,油菜种植也面临这一问题,在此背景下,扩大油菜种植面积和培育高产新品种已成为世界范围内公认的提高油菜籽总产量的有效途径。目前我国存在大量的盐碱土壤,如何对其加以利用成为作物生产亟待解决的问题。与改良盐碱土壤相比,培育适宜在盐碱地生长的耐盐碱新品种是更为经济有效的办法。因此,将培育耐盐碱的甘蓝型油菜品种作为新时期的育种目标具有重要的战略意义。本研究以甘蓝型油菜耐盐品系“2205”、敏盐品系“1423”及其衍生的82个重组自交系为研究材料,从耐碱性盐的QTL定位、耐碱性盐候选基因的挖掘与功能验证三个方面展开,取得了以下研究结果。1、甘蓝型油菜耐盐碱性指标评估利用水培法对甘蓝型油菜亲本及其82个重组自交系进行培养,生长至五叶期时,利用NaHCO₃模拟碱性盐胁迫处理,测量群体耐盐相关指标并对其做相关性分析,结果表明,在挑选耐盐指标时可优先考虑FWR（根鲜重）和SP（可溶性蛋白）;主成分分析的结果显示,主成分1、主成分2、主成分3的贡献率分别为25.65%、20.04%、15.52%,累计贡献率为61.21%;主成分1中SP的占比最大,其次是FWR;主成分2中SS（可溶性糖）占比最大,FWR次之;主成分3中EC（电导率）和SPAD（叶绿素含量）占比最大。这表明植株在遭受碱性盐胁迫时,会通过调节体内可溶性糖的含量以及根系的吸水能力来维持渗透压平衡,以此保持植株的正常形态以及生命特征。2、甘蓝型油菜耐碱性盐相关性状QTL定位及候选基因分析对亲本材料及其82个重组自交系在75 mmol/LNaHCO₃处理后的相关性状做统计分析,结果表明SPAD、RL、SP的变异系数相对较小,说明这些指标在群体中的离散程度较小;多为正态和偏正态分布,可以用于QTL分析。相关性分析表明STG与FWR和SP存在显著的负相关性,相关系数分别为-0.351**和-0.298**;SP与FWR、SPAD、SOD之间存在显著正相关性,与EC存在显著负相关,相关性系数分别为0.278**、0.237**、0.462**,-0.290**;FWR与SP（0.278**）、SOD（0.235**）、SS（0.248**）存在显著正相关性,与STG（-0.351**）存在显著负相关性。利用Win QTLcart 2.5软件对重组自交系群体性状进行QTL分析,结果显示共鉴定到12个主效QTL区间,主要分布在A03、A06、A09、A10、C03染色体上,其中,FWR定位得到4个QTL位点,RL定位得到1个QTL位点,SP定位得到4个QTL位点,SPAD定位得到3个QTL位点。贡献率在6.63%-27.19%之间,其中q SPAD-1位点贡献率最低,LOD值为2.850,q SPAD-3位点贡献率最高,LOD值为7.619。在A03染色体上鉴定得到的q SP-2（A03,71.4-75.8 c M）与q RL（A03,65.7-72.8 c M）位置接近,A09染色体上q SP-3（A09,87.8-95.3 c M）与q SPAD-1（A09,79.7-88.3 c M）位置最近。利用生物信息学技术在甘蓝型油菜数据库中对本章中定位得到的QTL区间进行blast比对,结果显示12个主效QTL区间共包含1563个基因,通过筛选前期本实验室提供的转录组数据中得差异基因位点,结合拟南芥数据库中的基因注释,确定了3个与耐盐碱相关的候选基因:BnaA06g13250D、BnaA03g25130D、BnaC03g28690D。3、耐碱性盐相关基因的克隆与功能验证利用同源重组的方法从两亲本中分别克隆得到了BnaA06g13250D、BnaA03g25130D、BnaC03g28690D基因,序列对比结果显示,在两亲本间克隆得到的基因均存在氨基酸序列差异,Bna13250-1423和Bna13250-2205之间有18个氨基酸差异,对于Bna25130-1423和Bna25130-2205存在3处氨基酸不同,BnaC03g28690D和Bna28690-1423序列完全一致,与BnaC03g28690D和Bna28690-1423相比,Bna28690-2205所编码的氨基酸序列在第2、114、136、144处存在差异。除此之外,基因编码的蛋白二三级结构均存在差异。对克隆得到的基因进行遗传转化,在野生型拟南芥（Wild type Clo-0,WT）中对基因进行功能验证。结果表明,在盐碱胁迫处理下,Bna13250-2205的过表达株系发芽率明显小于野生型,Bna25130-2205、Bna28690-2205基因的过表达株系的发芽率均高于野生型。正常处理条件下,与WT相比,Bna13250-2205根长更短,Bna25130-2205和Bna28690-2205基因的转基因过表达株系根长则无明显变化;在0.5mmol/L和1.0 mmol/L的碱性盐处理浓度下,与WT相比Bna25130-2205和Bna28690-2205基因过表达株系根长都无明显变化,但Bna13250-2205的转基因过表达株系与WT相比根长都明显下降。以上结果表明Bna25130-2205和Bna28690-2205可能参与植物芽期耐碱性的正调控,而Bna13250-2205负调控植物的芽期和苗期的耐盐碱性。更多还原

【Abstract】 Brassica napus L.is the most important cultivated type of rapeseed in China,With increasing of the world’s population,the available lands are gradually decreasing and the planting area of rapeseed was also facing this problem Under this background,expanding the planting area of rapeseed and cultivating high-yield varieties have become an effective way to increase the yield of rapeseed in the world.There are a lot of saline-alkali soils in our country,and how to make use of them has become an urgent problem.Compared with improving saline-alkali soil,it is more economical and effective to cultivate saline-alkali-tolerant varieties suitable for growing in saline-alkali land.Therefore,Cultivating the saline-alkali-tolerant rapeseed varieties was considered as an important breeding aim at the new period.In this study,a salt-tolerant line“2205”,a salt-sensitive line“1423”and 82 recombinant inbred lines derived from them were used as research materials.The following research results were obtained from three aspects:QTL mapping of alkali tolerant salt,isolation and functional verification of alkali tolerant salt candidate genes.1、Effects of alkaline stress on physiological indicators of B.napusThe two parents and 82 recombinant inbred liness were cultured by hydroponics.When they grew to the five-leaf stage,NaHCO₃ was used to simulate alkaline stress.The contribution rates of principal component 1,principal component 2,and principal component 3 are 25.65%,20.04%,and 15.52%,respectively,and the accumulated contribution rate was 61.21%.SP（soluble protein）accounted for the largest proportion in main component 1,followed by FWR;SS（soluble sugar）accounted for the largest proportion in main component 2,followed by FWR;EC（conductivity）and SPAD（chlorophyll content）accounted for the largest proportion in main component 3.This indicates that FWR（root fresh weight）and SP should be firstly selected as the salt tolerance indicators.2、QTL mapping of alkali tolerance-related traits in Brassica napus L.and candidate gene analysisThe parent material and 82 recombinant inbred lines were cultured by hydroponics,and phenotypic variation for all traits were analyzed after being treated with 75 mmol/L NaHCO₃.The results showed that the coefficients of variation of SPAD,RL and SP were relatively low,indicating that the degree of dispersion of these indicators is small in the population;and all traits showed continuous normal or near-normal distributions which can be used for QTL analysis.Correlation analysis showed that STG was significantly negatively correlated with FWR and SP and the correlation coefficients are-0.351**and-0.298**respectively;SP was significantly positively correlated with FWR,SPAD and SOD,while significantly negatively correlated with EC and the correlation coefficients are 0.278**,0.237**,0.462**and-0.290**respectively;FWR was significantly positively correlated with SP（0.278**）,SOD（0.235**）,and SS（0.248**）,while significantly negatively correlated with STG（-0.351**）.Win QTLcart 2.5 software was used to identify QTLs for all traits.The results showed that a total of 12 main QTL intervals were identified,mainly distributed on chromosomes A03,A06,A09,A10 and C03,including four QTLs for FWR,one QTL for RL,four QTLs for SP,three QTLs for SPAD.The phenotypic variance values（PVE）were ranged from 6.63%to 27.19%,of which q SPAD-1 has the lowest PVE with the LOD value 2.85 and q SPAD-3 has the highest PVE with the LOD value 7.62.q SP-2（A03,71.4-75.8 c M）identified on chromosome A03was close to q RL A03,65.7-72.8 c M),and q SP-3（A09,87.8-95.3 c M）was closest to q SPAD-1（A09,79.7-88.3 cM）on chromosome A09.Using bioinformatics technology to blast the QTL regions located in this chapter in the Brassica napus L.database,the results show that the 12 major QTL regions contain a total of 1563 genes.The differential gene loci were obtained and combined with the gene annotation in the Arabidopsis database,three candidate genes related to salt tolerance were identified:BnaA06g13250D,BnaA03g25130D,BnaC03g28690D.3、Cloning and functional verification of genes related to alkaline toleranceThe genes BnaA06g13250D,BnaA03g25130D,and BnaC03g28690D were cloned from the two parents by homologous recombination.The sequence comparison results showed that there were differences in the amino acid sequences of the cloned genes between the two parents.There are 18 amino acid differences.There are 3 amino acid differences between Bna25130-1423 and Bna25130-2205.The sequences of BnaC03g28690D and Bna28690-1423 are completely identical,136,144 there are differences.In addition,there are differences in the secondary and tertiary structures of proteins encoded by genes.The results showed that under alkaline stress,the germination rate of the Bna13250-2205 overexpression line was significantly lower than that of the wild type,and the germination rates of the Bna25130-2205and Bna28690-2205 gene overexpression lines were higher than those of the wild type.Under normal conditions,the root length of Bna13250-2205 was shorter than WT,but the root length of Bna25130-2205 and Bna28690-2205 overexpression lines of had not significant change compared with WT.Under the 0.5 mmol/L and 1.0 mmol/L NaHCO₃ treatment,the root length of Bna25130-2205 and Bna28690-2205 overexpression lines had no significant change,while the Bna13250-2205 overexpression lines exhibited significantly decrease compared with WT.The above results suggested that Bna25130-2205 and Bna28690-2205 may positively regulate the alkaline tolerance of plants at the bud stage,while Bna13250-2205 may negatively regulate the alkaline tolerance of plants at the bud and seedling stages.更多还原