【作者】 高必军；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 油菜是一种重要的油料作物，在世界各地除非洲少数地区以外，几乎都有栽培。油菜经济价值的提高首先需要改良油菜栽培品种，而油菜新品种的改良主要体现在油菜品质和产量的改良两个方面。在油菜品质改良方面，利用基因工程手段来改变油菜种子的化学组成成分及其含量是改良油菜品质的一个有效措施，其中，基因工程手段的一个关键技术就是调节控制优良基因的表达，而高等植物基因调控主要是在转录水平上进行的，受多种顺式作用元件和反式作用因子的相互协调作用。植物基因启动子是重要的顺式作用元件，是RNA聚合酶识别并与之结合，从而起始基因转录的一段DNA序列。因此，油菜组织特异性启动子的克隆和表达验证是一项重要工作。在油菜产量改良方面，由于油菜产量及与产量有关的其它许多农艺性状均表现为微效多基因控制的数量遗传，研究这些数量性状位点（QTL）在油菜基因组染色体上的位置和对性状表现影响的程度，具有积极现实的意义。近年来，随着数量遗传学和现代生物技术的发展，借助DNA分子标记和QTL作图，复杂的数量性状可剖分为若干离散的孟德尔因子所决定的组分，进而确定其在染色体上的位置及其效应大小。同时，应用分子标记辅助选择（MAS）和转基因等技术，使油菜产量的进一步提高成为可能。1．甘蓝型油菜napin基因启动子的克隆Napin是一类由多基因编码的种子贮藏蛋白，广泛存在于芸苔属植物的种子里，占种子蛋白总量的20％～30％。Napin基因是在ABA的影响下，以组织特异型方式表达。本实验根据GenBank中已经公布的napA全序列5′端非编码区设计引物，以甘蓝型油菜中双4号基因组DNA为模板，通过PCR扩增，获得的扩增产物克隆到pMD18-T载体并测序。结果表明，所克隆的扩增序列长度为1159bp，经比较分析，该序列与napA相比，只是有几个核苷酸差异，与其它已公布的napin基因启动子同源性也很高。该序列富含AT碱基，具有TATA-box、CAAT-box、G-box、GATA-box等高等植物启动子的序列特征。为了进一步验证其组织特异性表达功能，把它插入到pCAMBIA-1301载体，替代pCAMBIA-1301中GUS基因前的CaMV35S启动子，构建成GUS基因表达载体pC1301.N。把重组载体转化到农杆菌LBA4404中，感染ABA诱导的油菜种子，经GUS染色证明，该napin基因启动子具有种子特异性表达功能。2．甘蓝型油菜几个重要农艺性状的初步QTL定位利用杂交组合中双4号×H228 F₁，经8代自交，构建成RIL群体，在第8代时调查亲本和随机选择的142个RIL群体株系株高、一次有效分枝数、单株产量等11个农艺性状。RIL群体中11个农艺性状之间相关分析表明：单株产量与株高、一次有效分枝数、主花序长度、主花序有效角数、主花序角密度、角果长度、每角粒数、千粒重、单株有效角果总数呈极显著正相关。选用SSR引物对中双4号和H228间进行多态性检测，有152对引物在双亲间表现多态性，用这些引物分别对142个株系进行检测，构建了含有123个SSR分子标记、18个连锁群的连锁遗传图，总图距为3483.1cM，标记间平均距离为28.32cM，各标记在18个连锁群上的分布很不均匀，其中，第1、12、13、17连锁群上分布标记较多，其它连锁群上分布标记较少。根据RIL群体性状方差和SSR差异位点分析，该群体可以进行初步的遗传图谱构建和QTL定位，但各个性状的平均纯合度不太纯，还需进一步自交纯合，本次所获得遗传图谱和QTL位点将有一定误差。与Lowe等（2004）的遗传图谱比较，发现26个相同的标记位点，只占本实验标记位点的21.1％。11个农艺性状共检测到81个数量性状位点（QTLs）。其中，株高检测到4个QTLs，解释性状表型变异的10.3％～28.9％；一次有效分枝数检测到2个QTLs，解释性状表型变异的22.1％和47％；有效分枝部位检测到16个QTLs，解释性状表型变异的12.2％～51.8％；主花序长度检测到15个QTLs，解释性状表型变异的7.4％～26.6％；主花序有效角数检测到5个QTLs，解释性状表型变异的11.2％～25％；主花序角密度检测到1个QTLs，解释性状表型变异的17.3％；角果长度检测到12个QTLs，解释性状表型变异的24％～36.7％；每角粒数检测到2个QTLs，解释性状表型变异的9.6％和16.9％；千粒重检测到2个QTLs，解释性状表型变异的26％和13.7％；单株有效角果总数检测到11个QTLs，解释性状表型变异的14.8％～47.2％；单株产量检测到11个QTLs，解释性状表型变异的14.3％～32.8％。此外，还对GUS表达、启动子效率、遗传图距及影响因素、QTL定位的精确度等进行了讨论。更多还原

【Abstract】 Rapeseed is one of the most important oil crops. It is planted all over the worldexcept in a few areas of Africa. Increasing rapeseed economical value needs theimprovement of plant variety of rapeseed at first, while the improvement is embodiedin the two aspects of quality and yield of rapeseeds. In the aspect of qualityimprovement of rapeseeds, one of the effective ways is the application of geneticengineering in the change of chemical composition and its content. And one of keytechnology is to regulate the expression of the excellent gene. The gene regulation ofthe higher plant mainly happens in the transcription level and is affected by theinteraction of various cis-acting and trans-acting factors. The gene promoter is animportant cis-acting factor in plant, a DNA sequence which is identified andcombined by RNA polymerase and thus initiating the gene transcription. Therefore,the cloning and validating of organic specific promoter in rapeseed is an importanttask.In the improvement of the rapeseed yield, it is of positive and practicalsignificance to make research on these quantitative trait loci （QTL） in the position ofthe rapeseed genome and its extent of influence on the phenotype, because therapeseed yield and the many other yield-related agronomic traits are manifested asminor genes controlled quantitative inheritance. In recent year, along with thedevelopment of quantitative genetics and modem biotechnology, the complexquantitative traits could be separated to many independent Mendelian factors by DNAmolecular marker and QTL mapping and determine its position in genome and itseffect. Meanwhile, the application of techniques such as MAS, transgene, etc. makesit possible to further improve the rapeseeds.1. Cloning of the promoter of gene Napin in Brassica napus Napin,a variety of seed storage protein encoded by multi-gene, exists widely inseed of Brassica species, occupying 20%～30% of total protein in seed. In the affectof ABA, napin gene is tissue-specifically expressed.A pair of primers was designed according to 5’non-encoding region of the napAsequence reported in GenBank. With as the genomic DNA of Brassica napus"Zhongshuang 4 hao" as the template, the amplified products by PCR was cloned intopMD 18-T Vector and sequenced. The results indicated the cloned fragment contained1159bp. Through analysis, the fragment had only a few nucleotides differences withthe reported napA, and had high homology with the other reported napin genepromoter, too.The fragment is rich in AT nucleotides, with the sequence features of higher plant,such as TATA-box, CAAT-box, G-box, and GATA-box. Furthermore, to investigatethe tissue-specific expression function of the napin gene promoter, the fragment wasinserted into pCAMBIA-1301 vector, in place of the promoter CaMV35S in the upperstream of GUS in pCAMBIA-1301 vector, and the GUS gene expressing vector ofpC1301.N was constructed. The recombined vector was transformed intoagrobacterium LBA4404. The Brassica napus seeds via ABA inducement wereinfected by pC 1301.N/LB4404, and then were dyed by GUS. The result proved thatthe napin gene promoter was seed specifically expessed.2. Preliminery QTL of some Agronomically Import Traits in Brassica napusRIL population was constructed using the F₁ descendants of combinationzhongshuang No.4×H228 constructed via seventh inbred. The 11 agronomic traits,such as plant height, No. of effective 1st branches, yield per plant, etc. wereinvestigated from parent and random 142 F₈ RIL population plant series. Thecorrelation analysis of the 11 traits in RIL population showed that plant height, No. ofeffective 1st branches, length of main inflorenscence, effective siliques of maininflorenscence, density of main inflorenscence, length of silique, seed per sillique,1000 seed weight, total effective siliques per plant had significant positive correlationwith yield per plant.Polymorphisms between zhongshuang No.4 and H228 was examined by SSR primers. 152 primers Showed polymorphisms, and a linkage map containing 123markers, including 18 linkage groups was constructed. The total genetic distance ofthe map was 3483.1cM, and the average distance of two adjacent markers was28.32cM. The distribution of marker in 18 linkage groups was not even. Meanwhile,the first, twelfth, thirteenth, seventeenth linkage groups contained more marks, whilethe rest had fewer.According to the analysis of traits mean variance and SSR discrepancy situ inRIL, the constructing of genetic map and QTL could be processed preliminary. But,the average of traits were not homogeneous in some sort, the RIL need was purifiedunceasingly. The genetic map and QTL in this paper had error in some sort. Thecompare of genetic map between Lowe’s and ours showed 26 homology marker situ,which occupied 21.1% of the marker situ in the experiment.81 QTLs were detected for 11 agronomic traits. 4 QTLs were detected for plantheight, which explained 10.3%～28.9% of trait variance; 2 QTLs were detected forNo. of effective 1-st branches, which explained 22.1%～47% of trait variance; 16QTLs were detected for effective branches height, which explained 12.2%～51.8% oftrait variance; 15 QTLs were detected for length of main inflorenscence, whichexplained 7.4%～26.6% of trait variance; 5 QTLs were detected for effective siliquesof main inflorenscence, which explained 11.2%～25% of trait variance; 1 QTLs weredetected for density of main infiorenscence, which explained 17.3% of trait variance;12 QTLs were detected for length of silique, which explained 24%～36.7% of traitvariance; 2 QTLs were detected for seed per sillique, which explained 9.6% and16.9% of trait variance; 2 QTLs were detected for 1000 seed weight, which explained26%～13.7% of trait variance; 11 QTLs were detected for Total effective siliques perplant, which explained 14.8%～47.2% of trait variance; 11 QTLs were detected forplant height, which explained 14.3%～32.8% of trait variance.On the other hand, in this paper we also discussed on the expression of GUS, theefficiency of promoter, the genetic distance and effective factor, precision of QTL,etc.更多还原