甘蓝蜡质缺失基因BoGL4和BoGL1的克隆及功能分析

发布时间：2018-03-20 08:16

本文选题：甘蓝　切入点：表皮蜡质　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：结球甘蓝(Brassica oleracea L.var.capitata L.)简称甘蓝,起源于地中海沿岸,是十字花科芸薹属的一种重要蔬菜,在蔬菜周年供应和出口贸易中占有重要地位。普通结球甘蓝叶色多为绿色、深绿色或灰绿色,叶片及茎表皮覆盖有一层蜡质。蜡质缺失亮绿甘蓝由普通甘蓝突变而来,具有叶色亮绿美观等特点。本研究以两份甘蓝蜡质缺失突变体LD10和10Q-974为研究材料,对两份材料蜡质缺失突变基因进行了定位,并对LD10候选目的基因进行了克隆和功能验证。主要结果如下:1. 甘蓝蜡质缺失突变体LD10和10Q-974均具有叶片亮绿、商品性好的特点,且两者表皮蜡质晶体严重缺失,其中LD10蜡质缺失成分主要是C22-C30初级醇,10Q-974缺失成分主要是C29和C33烷烃。试配杂交组合结果显示,两甘蓝突变体材料的亮绿性状均具有较好的育种应用价值。2.以蜡质缺失突变体LD10和普通有蜡质芥蓝材料M36为亲本构建六世代群体,其中F_1群体叶面均有蜡质覆盖,F_2群体有蜡质和无蜡质植株的分离比例符合3:1,BC_1群体有蜡质和无蜡质植株的分离比例符合1:1,BC_2群体均表现为有蜡质性状,据此推断LD10的蜡质缺失性状由1对隐性基因(Bo GL4)控制。以同样的方法对10Q-974的蜡质缺失性状进行遗传分析,结果表明该突变体的蜡质缺失性状由1对显性基因(Bo GL1)控制。3.在连锁标记筛选、基因定位的基础上,将蜡质缺失突变体LD10的目的基因Bo GL4精细定位在1号染色体标记C01g SSR 147和C01g SSR 150之间,两标记间的物理距离是170kb,遗传距离为0.2c M,该定位区间内存在一个与拟南芥蜡质合成基因CER4同源的基因Bol013612,将其选定为Bo GL4的候选目的基因;将突变体10Q-974的目的基因Bo GL1定位于甘蓝8号染色体末端177kb的区间内,将定位区间内一个与拟南芥蜡质合成基因CER1同源的基因Bol018504确定为Bo GL1的候选目的基因。4.对Bol GL4和Bo GL1的候选目的基因进行克隆、测序,结果表明Bo GL4的候选目的基因Bol013612在g DNA上存在一个碱基的替换突变(A替换为G),该突变导致c DNA序列上六个碱基的插入;Bo GL1的候选目的基因Bol018504在g DNA和c DNA水平上均未发现碱基突变,排除了Bol018504是Bo GL1基因的可能性。5.利用拟南芥蜡质缺失突变体cer4-1为转化受体,对甘蓝Bo GL4的候选目的基因Bol013612进行了功能验证。结果表明转Bol013612基因的拟南芥突变体蜡质缺失性状得到了恢复;而转突变体LD10的Bol013612基因的拟南芥突变体蜡质缺失性状没有获得恢复,表明LD10的Bol013612基因存在功能缺失,这一实验结果进一步证明了LD10的蜡质缺失性状是由基因Bol013612突变造成的。
[Abstract]:Cabbage (Brassica oleracea L.var.capitata L.) referred to as cabbage, originated in the Mediterranean coast, is a kind of important vegetable Brassica, plays an important role in vegetable supply and export trade. The common cabbage leaf color is green, dark green or gray green, leaf and stem epidermis covered with a layer of wax. Wax light green cabbage cabbage by deletion mutations, has the characteristics of beautiful green leaves Shailiang. In this study, two accessions of Brassica waxy mutant LD10 and 10Q-974 as research materials, the two accessions waxy deletion mutation gene was located on LD10, and the candidate gene were cloned and verified. The main results are as follows cabbage: 1. waxy mutant LD10 and 10Q-974 have bright green leaves, good characteristics, and a serious lack of both epicuticular wax crystals, which is mainly composed of LD10 wax missing C22-C30 primary alcohols, 10Q-974 deletion composition is mainly C29 and C33 alkanes. Test hybrids showed that two cabbage mutant bright green characters have the breeding value of.2. good waxy mutant LD10 and common waxy Gai Lan material M36 were constructed in six generation population, the F_1 population had leaf waxy covering. The separation ratio of F_2 group and non waxy waxy plants with 3:1, BC_1 group has the separation ratio of wax and non wax plants with 1:1, BC_2 group showed waxy waxy trait, lack of traits inferred LD10 by 1 recessive genes (Bo GL4). In the same way as the lack of wax on the performance of 10Q-974 the genetic analysis results show that the wax loss characteristics of the mutant by 1 dominant genes (Bo, GL1).3. in screening markers, the foundation of gene mapping, the waxy deletion mutation Variant of LD10 gene Bo GL4 fine mapping of chromosome 1 between C01g SSR 147 and C01g SSR 150, the physical distance between the two markers is 170kb, the genetic distance was 0.2C M, the range of memory in a synthetic wax and Arabidopsis CER4 homolog gene Bol013612, which is selected as the candidate gene Bo GL4; Bo gene mutants of 10Q-974 GL1 interval is located in chromosome 8 of cabbage at the end of 177KB, will be located in this region with a synthetic CER1 gene homologous to the Arabidopsis waxy gene Bol018504 was identified as a candidate for GL1 based Bo to.4. on Bol GL4 and Bo GL1 of the candidate gene were cloned and sequenced. The results show that the substitution mutation candidate gene GL4 Bol013612 Bo with a base in G on DNA (A = G), the C DNA mutation in the sequence insertion of six nucleotides; candidate genes for Bol Bo GL1. In 018504 g DNA and C DNA levels were not found on the mutation of Bol018504 is.5., ruled out the possibility of Bo GL1 gene of Arabidopsis thaliana using waxy mutant cer4-1 as transformation receptor, the candidate gene Bol013612 of Brassica Bo GL4 function has been verified. The results show that the wax deletion of Bol013612 transgenic Arabidopsis mutant traits have been restored; deletion of Bol013612 gene of Arabidopsis thaliana mutant waxy traits and mutant LD10 has not been restored, the presence of LD10 showed that Bol013612 gene loss of function, the experiment results prove that the lack of waxy trait of LD10 is caused by a mutation of the Bol013612 gene.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S635

【参考文献】