【作者】 肖霞；

【导师】 殷家明； 柴友荣；

【作者基本信息】 西南大学 ， 作物遗传育种， 2010， 硕士

【摘要】 甘蓝型油菜(Brassica napus)黄籽与黑籽类型相比,具有种皮薄、含油量高、蛋白质含量高等优点,自问世以来,一直备受广泛关注。目前,黄籽甘蓝型油菜品种极少、种质资源有限,育种难度很大。培育遗传简单、性状稳定的黄籽甘蓝型油菜是油菜育种的重要目标之一。黄酮类化合物广泛存在于植物中,是植物重要的次生代谢产物,是十字花科等植物器官组织表面着色的重要成分。同时,黄酮类化合物也是公共苯丙烷途径核心分支途径即类黄酮途径的主要产物,对苯丙烷途径终产物——原花青素的合成起重要作用。在拟南芥(Arabidopsis thaliana)中,由于削弱了类黄酮途径,导致种皮颜色由野生型的深褐色变成黄色或灰褐色,这种突变体称透明种皮(transparent testa, tt)突变。目前,已鉴定出22个拟南芥透明种皮基因位点,其中大多数基因已被克隆。在拟南芥中,查尔酮异构酶(chalcone isomerase, CHI/TT5)催化6’-羟基查尔酮转变为5’-羟基黄烷酮,是类黄酮合成途径第二个关键酶,对花青素和原花青素的合成和积累起重要作用。TT8基因编码一个bHLH型正调控转录因子,它与TT2、TTG1形成一个三元复合物,调控类黄酮合成途径的“晚期”结构基因如DFR、BAN等的表达,从而调控原花青素、花青素苷的积累。本研究对反义BnTT5基因片段和反义BnTT8基因片段的转基因甘蓝型油菜植株及其后代进行了分析,结果如下：(1)甘蓝型油菜BnTT5反义转化植株的Southern杂交结果表明,外源反义BnTT5基因的构建物已成功整合到甘蓝型油菜基因组中。转基因后代种籽粒色与对照相比有一定变浅,说明BnTT5基因确实参与决定甘蓝型油菜等芸(?)属植物的种皮色素合成。对所有反义BnTT5转基因植株的花朵进行了观察,并没有发现与对照有什么差异,仍然是正常的金黄色,因此本研究所反义抑制的BnTT5靶基因到底与油菜花色形成有无关系或有多大关系,尚待研究。转基因后代植株形态特征和生长情况与CK植株无明显差异。(2)甘蓝型油菜BnTT8反义转化植株的Southern杂交结果说明反义BnTT8基因成功整合到甘蓝型油菜基因组中。通过半定量RT-PCR法检测结果表明,内源BnTT8基因的表达具有组织特异性,在转基因的花后35 d种籽中,该基因表达受到一定程度的抑制,但抑制程度较弱而且不稳定。GUS染色检测呈阳性的植株收获种籽的颜色与对照相比有明显变浅,虽然没有达到标准的金黄色性状,但已比较接近黄籽。所得到的反义BnTT8转基因后代植株主体形态特征与CK植株无明显差异,但是转基因种子普遍饱满度下降,粒形偏离球形,粒重由CK的3.15g下降到平均2.54g左右。这说明,BnTT8基因除了参与种皮色素合成以外,可能还参与调控了一些其它种子性状。(3)研究中发现,转基因植株中无论是T1和T2代转化植株的叶片GUS染色结果,还是T1代小孢子培养苗和T3植株的子叶和下胚轴的GUS染色鉴定结果,均是符合孟德尔遗传规律的。转基因后代能稳定存活,转入的外源基因和转基因性状修饰亦能稳定遗传。(4)反义BnTT5每个独立转基因株系的Southern杂交显示的外源基因杂交条带为1-2,对应花粉植株中多数GUS阳性和阴性的分离比均接近3：1,T3代植株的GUS阳性／阴性分离比接近15：1,符合孟德尔遗传的2对独立等位基因的分离比,表明普遍可能有2个转基因拷贝整合到受体株的基因组中,比前面Southern杂交结果显示的整个拷贝数偏多。但是,反义BnTT8每个独立转基因株系的Southern杂交显示的外源基因杂交条带为0-1,后代GUS染色结果普遍符合一对等位基因的分离比。因此,反义BnTT8转基因油菜后代的基本上普遍符合单拷贝整合,但反义BnTT5转基因油菜后代普遍符合双拷贝甚至更多拷贝插入,原因有待分析。(5)转反义BnTT5和BnTT8基因均能抑制内源基因的表达,但二者对靶基因的抑制程度不同。甘蓝型油菜的BnTT5基因家族成员数(约8个)远远比BnTT8的基因家族成员数(约2个)多,且成员间的序列差异性也要大得多。根据本研究的结果,对于甘蓝型油菜这种多倍物种来讲,反义RNA等基因沉默手段对于成员间序列高度相似的微小基因家族的抑制效果更理想,而对于成员间序列差异显著的复杂基因家族的抑制效果则较差。此外,本研究所分析的所有转基因后代中,内源基因转录本和目标性状均有相当程度的残留,而且表现出了株间、籽粒间和环境间的变异。因此今后对于甘蓝型油菜这种多倍体物种的转基因分子育种而言,需要更为强大的基因沉默手段,才能达到更理想的抑制内源基因家族表达和性状修饰的效果,而且需要注意转基因后代外源基因表达水平的变异和易受环境影响的特点。更多还原

【Abstract】 Since the yellow-seeded Brassica napus line was screened out, it has been paid much and extensive attention to. In rapeseed, the yellow seeds have many good properties compared to the black seeds, such as thinner seed coat, less seed coat pigment, lower meal fiber content, higher seed oil content and meal protein content, etc. Presently, the germplasm of yellow-seeded B. napus is narrow and there are few yellow-seeded varieties. It is one of the important objectives to breed yellow-seeded rapeseed lines with stably and simply inherited yellow seed trait.Flavonoids exist in plants ubiquitously. They are the important plant secondary metabolizing products and are the main pigment components in Cruciferous plants. In Arabidopsis thaliana, the weakening of the flavonoids metabolizing may result in the seed color changing from dark brown to yellow or grey-brown. Such mutants in seed color are defined as transparent testa (tt). Now,22 TT loci have been identified and many of the TT genes have been cloned in A. thaliana.Chalcone isoenzyme (CHI, TT5) is the second pivotal enzyme in flavonoid pigment biosynthesis pathway in A. thaliana. It plays an important role in the synthesis and accumulation of anthocyanins and proanthocyanidins. In A. thaliana, the TT8 gene encodes a positive regulative factor belonging to the bHLH transcription factors. It acts with TT2 and TTG1 to form a tri-complex and regulates the expression of the terminal structure genes DFR and BAN etc. Consequently, it dominates the accumulation of anthocyanins and proanthocyanidins.In the present study, the transgenic B. napus progenies with antisense suppression of BnTT5 and BnTT8 gene families were widely characterized. The results are as follows: (1) The DNA Southern blot results of the transgenic accessions of the antisense BnTT5 suppression showed that the antisense BnTT5 T-DNA segment had been integrated into the B. napus acceptor genome successfully. Transgenic progeny seeds showed a trend of lightening when compated with non-transgenic CK, indicating that BnTT5 genes practically participate in determining the biosynthesis of seed coat pigments in Brasica species like rapeseed. The flowers of all BnTT5-antisense transgenic progenies showed little change, with still normal golden color. So whether the target BnTT5 genes antisense-suppressed in this study have any relations to rapeseed flower color formation deserves further study. Most other morphological traits and growth have no distict difference from the CK.(2) The DNA southern blot results of the transgenic accessions of the antisense BnTT8 suppression indicated that the antisense BnTT8 T-DNA segment had been integrated into the B. napus acceptor genome successfully. Semi-quantitative RT-PCR detection showed that the internal BnTT8 transcription had tissue-specificity. It showed some degree of suppression in the transgenic seeds of 35 d after flowering as compared with the CK, but the inhibition degree is weak and varied. The seeds of GUS-staining positive plants showed obvious lightening when compared with the CK. Though the transgenic seeds were not standar golden yellow, they were more yellowish than as black types. Most morphological traits of BnTT8-antisense transgenic progenies and growth have no distict difference from the CK, but the transgenic seeds generally showed decrease in seed-filling degree, with seeds deviated from standard ball-shape and decrease of 1000-seed weight from 3.15 g to 2.54 g on average. This study indicated that the BnTT8 genes might also participate in determination of some other seed traits besides seed pigmentation.(3) Results of GUS-staining of leaves of transgenic T1 and T2 plants, of embryos of pollen culture derived from T1 plants, and cotyledons and hypocotyls of T3 plants all showed that the transgenic plants conformed to Mendelian inheritance. Transgenic progenvies could successively survive and the transgenes together with the transgenic traits modifications could also be stably inherited.(4) In Southern detection, each independent transgenic line with antisense suppression of BnTT5 showed 1-2 hybridization bands. In most cases, the pollen plants from corresponding lines showed segregation ratios of GUS staining positive over negative reactions were near to 3:1. The T3 plants showed a ratio near 15:1 of GUS positive over negative reaction. The results conform to the segregion retios of 2 independent alleles, implying that possibly there were about 2 transgenes integraged into the genome of most antisense BnTT5-transformed lines, which were more that the Southern band numbers. In Southern detection, each independent transgenic line with antisense suppression of BnTT8 showed 0-1 hybridization band, and the GUS staining of the progenies conform to the segregation ratios of one independent alle. Hence, antisense BnTT8-transformed lines generally showed single-copy integration of the foreign construct, but antisense BnTT85-transformed lines generally showed double-copy integration of the foreign construct. The reason needs further study.(5) Antisense transformations of BnTT5 and BnTT8 both showed inhibition of the target genes, but they showed difference in suppression degrees. BnTT5 gene family has much more numbers (about 8) than BnTT8 gene family (about 2), and sequence divergence is also much higher. According to the results of this study, for polyploidy species like B. napus, gene silencing methodologies like antisense RNA are more effective on suppression of mini gene family containing members with high sequence similarities and less effective on gene family containing members with distinct sequence divergence. Furthermore, in this study, all transgenic progenies showed considerable remaining of target gene expression and target traits, and variations among plants, seeds and environments were also considerable. So in the future in transgenic molecular breeding for polyploid species like rapeseed, stronger gene silencing tools are needed to reach more optimal effect of suppressing internal gene expression and trait modification, and cautions should also be paid on the variations of transgene expression and environmental influence.更多还原