家蚕新突变体淡红卵re~p的定位分析

发布时间：2018-08-05 12:47

【摘要】：家蚕(Silkworm,Bombyx mori)是一种完全变态的昆虫,以卵繁殖,可以说卵是家蚕生命周期中第一个阶段,由此看出卵的研究有着非常特殊的价值。在家蚕饲养的过程中,因自然因素和人为因素的影响,在原有的卵色基础上,产生了许多卵色突变体,因其卵色种类繁多,且易于检测和识别,是研究遗传学的绝好材料。淡红卵突变体(pale red egg,re~p)是我们在品种选育工作中发现的一种新的卵色突变体,该家蚕品种刚产下的卵呈黄白色,在产后大约40 h开始变色,最终呈现淡红色。遗传分析表明,该突变性状由一对隐性基因控制,与红卵突变基因(re)可能为等位基因。本研究在此基础上通过利用SSR多态性标记和图位克隆技术对该突变基因进行连锁及精细定位分析,构建连锁图谱,进一步采用半定量、定量及克隆测序对定位区间内候选基因进行筛选和功能上的初步分析,以期找到引起该突变性状的目的基因,主要结果如下:一、突变体(re~p)的经典遗传学分析将淡红卵(re~p)与正常型黑卵(p50)进行杂交,结果显示:无论正反交,F1所产卵的卵色均表现正常型;F2所产卵的卵色出现分离现象,有正常型和淡红卵两种表型,其分离比为3:1;以正常型作为回交亲本,与正反交F1进行回交,回交后代均表现为正常型;以淡红卵作为回交亲本,与正反交F1进行回交,回交后代表现出正常型和淡红卵两种表型,其分离比为1:1。由此可见,re~p由1个隐性基因控制,且在常染色体上。将re~p与红卵(re)进行杂交,以re作为母本,F1所产卵均为红色卵,F1进行自交,F2所产卵均为红色卵,未出现卵色分离,F2再进行自交,自交后代F3卵色出现分离表现出红卵、淡红卵及其过渡色,但未出现明显的分离比;以re~p作为母本,F1所产卵均为淡红卵,F1进行自交,F2所产卵均为红卵,F2再进行自交,自交后代F3卵色出现分离表现出红卵、淡红卵及其过渡色,但未出现明显的分离比。由此可见,淡红卵与红卵为等位基因,还有母性影响表现。二、突变基因(re~p)的连锁及定位分析构建P1、P2、F1、F2、BC1F以及BC1M群体,用亲本P1、F1和P2筛选获得了家蚕28个连锁群上的SSR多态性标记,然后用BC1F群体中11个产淡红卵的突变体和11个产黑卵的正常个体来做连锁分析,结果表明re~p基因位于第5连锁群;用1087个F2个体和BC1M群体中68个突变个体进行精细定位分析。结果显示,根据筛选出的多态性SSR分子标记,最终将re~p基因定位于S2674-N53与S2674-N21两个分子标记之间,两标记间相距0.39 cM,物理距离约为370 kb,范围内有14个候选基因。三、候选基因的筛选及分析在定位分析的基础上,以野生型与突变型不同卵期的蚕卵为材料对区间内的14个候选基因进行qRT-PCR分析,来构建14个候选基因在C1(H)和re~p中各组不同卵期的表达谱;然后对部分候选基因(BGIBMGA003693、BGIBMGA003501、BGIBMGA003500、BGIBMGA003694、BGIBMGA003695、BGIBMGA003499)的ORF序列及BGIBMGA003694和BGIBMGA003695的3’端和5’端进行克隆测序,6个基因的ORF和2个基因的3’端和5’端在野生型C1(H)与突变型re~p中并无差异。四、MFS基因的结构分析MFS基因编码超家族转运蛋白(major facilitator superfamily),是re突变体产生的突变基因。通过遗传分析,红卵表型相对淡红卵表型为不完全显性,两者关系密切,且MFS基因位于定位区间内。故以野生型C1(H)和re~p突变体cDNA为模板,对MFS基因的CDS进行克隆测序,结果发现相较于C1(H),在re~p突变体中MFS基因第6号外显子上一段59 bp的序列被另一段14 bp的序列所替换,其他序列未发生变化。分析替换序列发现,这段序列只是影响了替换部分氨基酸的数目,替换位点前后氨基酸序列均未受到影响,没有改变编码蛋白的跨膜结构。通过qRT-PCR分析MFS基因在野生型C1(H)、re~p和re中组织表达谱和不同卵期表达谱,结果发现该基因在C1(H)和re~p的头部、表皮、脂肪体、后部丝腺、精巢中表达量较高,在气管和卵巢中表达量较低,且C1(H)和re~p没有显著性差异,然而在re所有组织中表达量很低几乎不表达。与组织表达谱一样,MFS基因在C1(H)、re~p和re不同卵期时的荧光定量PCR结果同样显示,MFS基因在野生型C1(H)和re~p突变体中表达量无显著差异,而在re突变体中表达量明显偏低,由此证明MFS基因是引起re突变表型的关键基因,是否是引起re~p突变性状的关键基因还有待进一步验证。上述研究确定了淡红卵突变体的定位区间,对区间内基因也进行了初步分析,为能找到突变体产生的突变基因,还需对候选基因进行深入的研究。
[Abstract]:Silkworm (Bombyx mori) is a kind of complete metamorphosis insect with egg reproduction. It can be said that egg is the first stage of the life cycle of the silkworm. It is found that the study of egg has a very special value. In the process of silkworm rearing, many egg color mutations have been produced on the basis of the original egg color on the basis of the natural and human factors. Body, because of its variety of egg color and easy to detect and identify, is a great material to study genetics. The pale red egg (re~p) is a new egg color mutant that we found in the breeding of varieties. The eggs of the silkworm breed are yellow white, and begin to change color after 40 h after postpartum. The analysis showed that the mutation was controlled by a pair of recessive genes, and the mutant gene of the red egg (RE) might be a allele. On the basis of this study, the mutation gene was linked and refined by using SSR polymorphism marker and map cloning technique, and the linkage map spectrum was constructed, and semi quantitative, quantitative and cloned sequencing were used. In order to find the target genes that cause the mutation, the main results are as follows: first, the classic genetic analysis of the mutant (re~p) crosses the light red egg (re~p) with the normal type of black egg (P50). The results show that the egg color of the egg produced by F1 is normal regardless of the positive and negative cross. The egg color of the egg produced by F2 appeared as the normal type and the light red egg of two phenotypes, and its separation ratio was 3:1; the normal type was used as a backcross parent, the backcross F1 was back cross, the backcross offspring all showed normal type; the light red egg was used as a backcross parent, and the backcross F1 was backcrossed, and the back cross showed two forms of normal and light red eggs. The separation ratio is 1:1., and re~p is controlled by 1 recessive genes, and on the autosomes, the re~p and the red egg (RE) are hybridized with re as the mother parent. The eggs of F1 are all red eggs, F1 is the red egg, the eggs of F2 are all red eggs, the egg color is not separated, the F2 is then self cross, the F3 egg color of the self cross offspring shows the red eggs, and the F3 ovum appearance of the self bred offspring shows the red egg, light, and light, the F3 ovum appearance of the offspring shows the red ovum, light, light and light eggs, light and light eggs, light eggs, light eggs, light eggs, light eggs, light eggs, light eggs, light eggs, light eggs, light eggs, light eggs, light eggs of the offspring F3 ovum color appearing in the offspring Red eggs and their transition colors, but there is no obvious separation ratio; re~p as the mother, the eggs of F1 are all light red eggs, F1 is inbred, F2 spawns are red eggs, F2 is then self inbred, and self cross offspring F3 egg color separation shows red eggs, light red eggs and their transition color, but there is no obvious separation ratio. So it can be seen that the pale red egg and the red egg are the same. Two, the linkage and location of mutant gene (re~p) and location analysis were used to construct P1, P2, F1, F2, BC1F and BC1M populations. The SSR polymorphic markers on the 28 chain of silkworm were obtained by screening the parent P1, F1 and P2, and then using 11 light red eggs producing mutants in the BC1F group and 11 normal individuals producing black eggs. The results showed that the re~p gene was located in the fifth linkage group, with 1087 F2 individuals and 68 mutant individuals in BC1M population. The results showed that the re~p gene was located between the two molecular markers of S2674-N53 and S2674-N21, and the two markers were 0.39 cM, and the physical distance was about 370. KB, there are 14 candidate genes. Three, the selection and analysis of candidate genes, based on the location analysis, use the wild type and mutant ovum eggs as the material to qRT-PCR analysis of 14 candidate genes in the interval, to construct the expression profiles of 14 candidate genes in C1 (H) and re~p, and then to some candidates. The ORF sequence of the gene (BGIBMGA003693, BGIBMGA003501, BGIBMGA003500, BGIBMGA003694, BGIBMGA003695, BGIBMGA003499) and the 3 'and 5' ends of BGIBMGA003694 and BGIBMGA003695 were cloned and sequenced. The 3 'end and 5' ends of the 6 genes were not different in the wild type C1 (H) and the mutant type. Four, the structural component of the gene. The MFS gene encoding superfamily transporter protein (major facilitator superfamily) is a mutant gene produced by the re mutant. Through genetic analysis, the phenotype of the erythrophenotypes relative to the red egg is not completely dominant, and the relationship is close, and the MFS gene is located in the location range. Therefore, the wild type C1 (H) and re~p mutant cDNA are used as a template for MFS gene CDS. The results showed that the sequence of 59 BP in the MFS gene sixth exon of MFS gene was replaced by another 14 BP sequence in the re~p mutant, and the other sequences did not change in the re~p mutant. The sequence of substitution found that the sequence only affected the number of the substituted amino acids, and the amino acid sequence before and after the replacement site was not. The transmembrane structure of the encoding protein was not changed. The expression profiles of MFS gene in the wild type C1 (H), re~p and re were analyzed by qRT-PCR. The results showed that the gene expressed in the head, the epidermis, the fat body, the posterior silk gland, the spermary, the spermary, the ovary and the ovary of the wild type C1 (H) and re~p, and the low expression in the trachea and ovary, and C1 (H). There was no significant difference with re~p, but the expression was very low in all re tissues. As with tissue expression, the MFS gene in C1 (H), re~p and re at different egg stages showed that the amount of MFS gene in the wild type C1 (H) and re~p mutants had no significant difference, but the expression in the mutant was obviously biased. It has been proved that the MFS gene is the key gene that causes the mutant phenotype of re. Whether it is the key gene that causes the mutation of re~p is still to be further verified. Carry out an in-depth study.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S881.2

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