發(fā)布時間：2018-03-20 08:16

  本文選題：甘藍(lán)　切入點：表皮蠟質(zhì)　出處：《華中農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：結(jié)球甘藍(lán)(Brassica oleracea L.var.capitata L.)簡稱甘藍(lán),起源于地中海沿岸,是十字花科蕓薹屬的一種重要蔬菜,在蔬菜周年供應(yīng)和出口貿(mào)易中占有重要地位。普通結(jié)球甘藍(lán)葉色多為綠色、深綠色或灰綠色,葉片及莖表皮覆蓋有一層蠟質(zhì)。蠟質(zhì)缺失亮綠甘藍(lán)由普通甘藍(lán)突變而來,具有葉色亮綠美觀等特點。本研究以兩份甘藍(lán)蠟質(zhì)缺失突變體LD10和10Q-974為研究材料,對兩份材料蠟質(zhì)缺失突變基因進(jìn)行了定位,并對LD10候選目的基因進(jìn)行了克隆和功能驗證。主要結(jié)果如下:1. 甘藍(lán)蠟質(zhì)缺失突變體LD10和10Q-974均具有葉片亮綠、商品性好的特點,且兩者表皮蠟質(zhì)晶體嚴(yán)重缺失,其中LD10蠟質(zhì)缺失成分主要是C22-C30初級醇,10Q-974缺失成分主要是C29和C33烷烴。試配雜交組合結(jié)果顯示,兩甘藍(lán)突變體材料的亮綠性狀均具有較好的育種應(yīng)用價值。2.以蠟質(zhì)缺失突變體LD10和普通有蠟質(zhì)芥藍(lán)材料M36為親本構(gòu)建六世代群體,其中F_1群體葉面均有蠟質(zhì)覆蓋,F_2群體有蠟質(zhì)和無蠟質(zhì)植株的分離比例符合3:1,BC_1群體有蠟質(zhì)和無蠟質(zhì)植株的分離比例符合1:1,BC_2群體均表現(xiàn)為有蠟質(zhì)性狀,據(jù)此推斷LD10的蠟質(zhì)缺失性狀由1對隱性基因(Bo GL4)控制。以同樣的方法對10Q-974的蠟質(zhì)缺失性狀進(jìn)行遺傳分析,結(jié)果表明該突變體的蠟質(zhì)缺失性狀由1對顯性基因(Bo GL1)控制。3.在連鎖標(biāo)記篩選、基因定位的基礎(chǔ)上,將蠟質(zhì)缺失突變體LD10的目的基因Bo GL4精細(xì)定位在1號染色體標(biāo)記C01g SSR 147和C01g SSR 150之間,兩標(biāo)記間的物理距離是170kb,遺傳距離為0.2c M,該定位區(qū)間內(nèi)存在一個與擬南芥蠟質(zhì)合成基因CER4同源的基因Bol013612,將其選定為Bo GL4的候選目的基因;將突變體10Q-974的目的基因Bo GL1定位于甘藍(lán)8號染色體末端177kb的區(qū)間內(nèi),將定位區(qū)間內(nèi)一個與擬南芥蠟質(zhì)合成基因CER1同源的基因Bol018504確定為Bo GL1的候選目的基因。4.對Bol GL4和Bo GL1的候選目的基因進(jìn)行克隆、測序,結(jié)果表明Bo GL4的候選目的基因Bol013612在g DNA上存在一個堿基的替換突變(A替換為G),該突變導(dǎo)致c DNA序列上六個堿基的插入;Bo GL1的候選目的基因Bol018504在g DNA和c DNA水平上均未發(fā)現(xiàn)堿基突變,排除了Bol018504是Bo GL1基因的可能性。5.利用擬南芥蠟質(zhì)缺失突變體cer4-1為轉(zhuǎn)化受體,對甘藍(lán)Bo GL4的候選目的基因Bol013612進(jìn)行了功能驗證。結(jié)果表明轉(zhuǎn)Bol013612基因的擬南芥突變體蠟質(zhì)缺失性狀得到了恢復(fù);而轉(zhuǎn)突變體LD10的Bol013612基因的擬南芥突變體蠟質(zhì)缺失性狀沒有獲得恢復(fù),表明LD10的Bol013612基因存在功能缺失,這一實驗結(jié)果進(jìn)一步證明了LD10的蠟質(zhì)缺失性狀是由基因Bol013612突變造成的。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S635

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