家蠶新突變體淡紅卵re~p的定位分析
發(fā)布時間：2018-08-05 12:47
【摘要】：家蠶(Silkworm,Bombyx mori)是一種完全變態(tài)的昆蟲,以卵繁殖,可以說卵是家蠶生命周期中第一個階段,由此看出卵的研究有著非常特殊的價值。在家蠶飼養(yǎng)的過程中,因自然因素和人為因素的影響,在原有的卵色基礎(chǔ)上,產(chǎn)生了許多卵色突變體,因其卵色種類繁多,且易于檢測和識別,是研究遺傳學的絕好材料。淡紅卵突變體(pale red egg,re~p)是我們在品種選育工作中發(fā)現(xiàn)的一種新的卵色突變體,該家蠶品種剛產(chǎn)下的卵呈黃白色,在產(chǎn)后大約40 h開始變色,最終呈現(xiàn)淡紅色。遺傳分析表明,該突變性狀由一對隱性基因控制,與紅卵突變基因(re)可能為等位基因。本研究在此基礎(chǔ)上通過利用SSR多態(tài)性標記和圖位克隆技術(shù)對該突變基因進行連鎖及精細定位分析,構(gòu)建連鎖圖譜,進一步采用半定量、定量及克隆測序?qū)Χㄎ粎^(qū)間內(nèi)候選基因進行篩選和功能上的初步分析,以期找到引起該突變性狀的目的基因,主要結(jié)果如下:一、突變體(re~p)的經(jīng)典遺傳學分析將淡紅卵(re~p)與正常型黑卵(p50)進行雜交,結(jié)果顯示:無論正反交,F1所產(chǎn)卵的卵色均表現(xiàn)正常型;F2所產(chǎn)卵的卵色出現(xiàn)分離現(xiàn)象,有正常型和淡紅卵兩種表型,其分離比為3:1;以正常型作為回交親本,與正反交F1進行回交,回交后代均表現(xiàn)為正常型;以淡紅卵作為回交親本,與正反交F1進行回交,回交后代表現(xiàn)出正常型和淡紅卵兩種表型,其分離比為1:1。由此可見,re~p由1個隱性基因控制,且在常染色體上。將re~p與紅卵(re)進行雜交,以re作為母本,F1所產(chǎn)卵均為紅色卵,F1進行自交,F2所產(chǎn)卵均為紅色卵,未出現(xiàn)卵色分離,F2再進行自交,自交后代F3卵色出現(xiàn)分離表現(xiàn)出紅卵、淡紅卵及其過渡色,但未出現(xiàn)明顯的分離比;以re~p作為母本,F1所產(chǎn)卵均為淡紅卵,F1進行自交,F2所產(chǎn)卵均為紅卵,F2再進行自交,自交后代F3卵色出現(xiàn)分離表現(xiàn)出紅卵、淡紅卵及其過渡色,但未出現(xiàn)明顯的分離比。由此可見,淡紅卵與紅卵為等位基因,還有母性影響表現(xiàn)。二、突變基因(re~p)的連鎖及定位分析構(gòu)建P1、P2、F1、F2、BC1F以及BC1M群體,用親本P1、F1和P2篩選獲得了家蠶28個連鎖群上的SSR多態(tài)性標記,然后用BC1F群體中11個產(chǎn)淡紅卵的突變體和11個產(chǎn)黑卵的正常個體來做連鎖分析,結(jié)果表明re~p基因位于第5連鎖群;用1087個F2個體和BC1M群體中68個突變個體進行精細定位分析。結(jié)果顯示,根據(jù)篩選出的多態(tài)性SSR分子標記,最終將re~p基因定位于S2674-N53與S2674-N21兩個分子標記之間,兩標記間相距0.39 cM,物理距離約為370 kb,范圍內(nèi)有14個候選基因。三、候選基因的篩選及分析在定位分析的基礎(chǔ)上,以野生型與突變型不同卵期的蠶卵為材料對區(qū)間內(nèi)的14個候選基因進行qRT-PCR分析,來構(gòu)建14個候選基因在C1(H)和re~p中各組不同卵期的表達譜;然后對部分候選基因(BGIBMGA003693、BGIBMGA003501、BGIBMGA003500、BGIBMGA003694、BGIBMGA003695、BGIBMGA003499)的ORF序列及BGIBMGA003694和BGIBMGA003695的3’端和5’端進行克隆測序,6個基因的ORF和2個基因的3’端和5’端在野生型C1(H)與突變型re~p中并無差異。四、MFS基因的結(jié)構(gòu)分析MFS基因編碼超家族轉(zhuǎn)運蛋白(major facilitator superfamily),是re突變體產(chǎn)生的突變基因。通過遺傳分析,紅卵表型相對淡紅卵表型為不完全顯性,兩者關(guān)系密切,且MFS基因位于定位區(qū)間內(nèi)。故以野生型C1(H)和re~p突變體cDNA為模板,對MFS基因的CDS進行克隆測序,結(jié)果發(fā)現(xiàn)相較于C1(H),在re~p突變體中MFS基因第6號外顯子上一段59 bp的序列被另一段14 bp的序列所替換,其他序列未發(fā)生變化。分析替換序列發(fā)現(xiàn),這段序列只是影響了替換部分氨基酸的數(shù)目,替換位點前后氨基酸序列均未受到影響,沒有改變編碼蛋白的跨膜結(jié)構(gòu)。通過qRT-PCR分析MFS基因在野生型C1(H)、re~p和re中組織表達譜和不同卵期表達譜,結(jié)果發(fā)現(xiàn)該基因在C1(H)和re~p的頭部、表皮、脂肪體、后部絲腺、精巢中表達量較高,在氣管和卵巢中表達量較低,且C1(H)和re~p沒有顯著性差異,然而在re所有組織中表達量很低幾乎不表達。與組織表達譜一樣,MFS基因在C1(H)、re~p和re不同卵期時的熒光定量PCR結(jié)果同樣顯示,MFS基因在野生型C1(H)和re~p突變體中表達量無顯著差異,而在re突變體中表達量明顯偏低,由此證明MFS基因是引起re突變表型的關(guān)鍵基因,是否是引起re~p突變性狀的關(guān)鍵基因還有待進一步驗證。上述研究確定了淡紅卵突變體的定位區(qū)間,對區(qū)間內(nèi)基因也進行了初步分析,為能找到突變體產(chǎn)生的突變基因,還需對候選基因進行深入的研究。
[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.
【學位授予單位】：江蘇科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S881.2
本文編號：2165841
[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.
【學位授予單位】：江蘇科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S881.2
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