利用MAS技术选育耐氟且无鳞毛家蚕新品种及AcMNPV敏感性基因的定位分析
发布时间：2019-06-06 02:30
【摘要】：随着社会经济的发展,空气污染在许多领域都是一个大问题。氟化物是污染空气的重要组成部分,它不仅对人体有害,还污染树木和桑蚕,危害着蚕桑产业。经研究表明家蚕耐氟性主要由显性主效基因(Dtf)控制着,并已利用SSR标记将其定位于家蚕第12连锁群。在我们的试验中菁松亲本是对氟敏感的,当喂食浸渍50 mg/kg NaF的桑叶时就会产生中毒症状,当喂食浸渍100 mg/kg NaF的桑叶时,幼虫全部死亡。而带有抗性基因的个体喂食浸渍200 mg/kg NaF的桑叶时几乎不产生中毒症状。家蚕是鳞翅目的代表,它的翅膀的主要特征是上面覆盖有一层鳞毛。在制种过程中会有大量的鳞毛脱落,飘散在空气中而成微尘,污染工作环境,危害制种工作人员的健康。蚕业学者通过资源考察与采集发现了它的突变体(成虫翅无鳞毛),经遗传分析,发现该性状是由隐性基因(nlw)控制的,并通过分子标记技术将其精细定位。本实验以菁松为轮回亲本,先后与U13和T6品种杂交。首先以菁松品种为母本,T6品系为父本杂交,然后以F1代为母本与U13品系杂交,后代BC1再与菁松母本回交,产生的下一代的BC2代中筛选经济性状最好的雄性个体作为父本与菁松母本回交。在每一次回交后代中,都用与家蚕耐氟性基因Dtf连锁的SSR、STS标记(S1214和S121201)以及与无鳞毛翅基因(nlw)连锁的分子标记(S1305和cash2p)来检测雄蛾子基因型。如果任一标记表现为菁松的纯合型,则该个体的后代予以淘汰,只留S1214和S121201位点表现为菁松与T6的杂合型并且在S1305和cash2p位点表现为菁松和U13杂合型的个体的后代,采用这种检测方法连续回交至BC6代。之后通过自交得到的群体喂食被200 mg/kg NaF污染的桑叶,然后存活下来个体选择无鳞毛翅且经济性状良好的家蚕被选择出来,自交产生下一代BC_6F_3。采用分子标记选出那些在Dtf和nlw位点纯合的个体。这样,具有良好性状,高耐氟性并无鳞毛的家蚕产生了。苜蓿银纹夜蛾核型多角体病毒(Autographed California multiple nucleopolyhedrovirus)作为杆状病毒的代表种,能够感染许多宿主。研究发现,通过注射接种的方法,重组AcMNPV可以感染部分家蚕品种,且家蚕对AcMNPV的感染性由隐性单基因(susceptible to AcMNPV,sav)控制。以家蚕不感染品种C108和感染品种P50为亲本组配正反交群体,并获得回交群体(P50×C108)×P50和P50×(P50×C108),记作BC1F和BC1M。在家蚕饲养过程中将分离群体的五龄幼虫注射含有GFP标记的重组AcMNPV病毒,并喂养浸渍有抗生素的桑叶3天后用荧光显微镜区观察。有绿色荧光的个体为感染个体,没有绿色荧光的个体为不感染个体。根据家蚕基因组信息,设计STS分子标记对sav基因进行定位分析,将sav基因定位在第3连锁群,绘制的遗传连锁图的遗传距离为48.7cM,sav基因位于37.6cM处,T0399和T03147标记与sav基因的距离最近,分别是5.1和0.4cM。
[Abstract]:With the development of social economy, air pollution is a big problem in many fields. Fluoride is an important part of polluted air, it is not only harmful to human body, but also pollutes trees and silkworm, and endangers sericulture industry. The results showed that the fluorine tolerance of Bombyx mori was mainly controlled by the dominant major gene (Dtf), and it had been mapped to the 12th linkage group of Bombyx mori by SSR markers. In our experiment, the parents of Pinus tabulaeformis were sensitive to fluorine, and the larvae died when fed with mulberry leaves soaked with 50 mg/kg NaF mulberry leaves, and when fed with mulberry leaves soaked with 100 mg/kg NaF, all the larvae died. However, individuals with resistance genes hardly had toxic symptoms when they were fed mulberry leaves soaked with 200 mg/kg NaF. Bombyx mori is the representative of Lepidoptera. The main feature of its wings is that it is covered with a layer of scaly hair. In the process of seed production, a large number of scales and hairs will fall off, drift into dust in the air, pollute the working environment, and endanger the health of seed production staff. Sericulture scholars found its mutant (adult wing without scaly hair) through resource investigation and collection. Through genetic analysis, it was found that the trait was controlled by recessive gene (nlw), and its fine mapping was carried out by molecular marker technique. In this experiment, Pinus elliottii was used as reincarnation parent and crossed with U13 and T6 varieties. First, the cyanine pine variety was used as the female parent, T6 strain as the male parent, then F1 generation as the female parent and U13 strain, and the progeny BC1 was backcrossed with the female parent of the juniper pine strain, and then the F1 generation was used as the female parent to cross with the U13 strain. In the next generation of BC2 generation, the male individuals with the best economic traits were selected as the male parent and the female parent of Pinus elliottii as backcross. In each backcross progenies, SSR,STS markers linked to fluorine-tolerant genes Dtf (S1214 and S121201) and molecular markers (S1305 and cash2p) linked to scalyless wing gene (nlw) were used to detect male moths. If any marker shows the homozygous type of cyanine pine, the offspring of the individual will be eliminated, leaving only the progenies of S1214 and S121201 loci as hetero-type of cyanine pine and T6 and the progenies of S1305 and cash2p loci showing cyanine pine and U13 heterozygous type. The detection method was used to backcross to BC6 generation. After that, the population obtained by self-cross fed mulberry leaves contaminated with 200 mg/kg NaF, and then survived, the silkworm, which had no scaly wings and good economic characters, was selected, and the next generation of BC_6F_3. was produced by self-cross. Molecular markers were used to select individuals who were homozygous at Dtf and nlw loci. In this way, silkworm (Bombyx mori) with good traits and high fluorine tolerance without scaly hair was produced. The nuclear polyhedrosis virus (Autographed California multiple nucleopolyhedrovirus), as a representative species of baculovirus, can infect many hosts. It was found that the recombinant AcMNPV could infect some Bombyx mori varieties by injection and vaccination, and the infection of AcMNPV to Bombyx mori was controlled by recessed single gene (susceptible to AcMNPV,sav. Bombyx mori non-infected variety C108 and infected variety P50 were used as parents to match positive and negative cross population, and backcross population (P50 脳 C108) 脳 P50 脳 (P50 脳 C108) was obtained, which was recorded as BC1F and BC1M.. The fifth instar larvae of Bombyx mori were injected with recombinant AcMNPV virus containing GFP markers during the feeding process, and the mulberry leaves soaked with antibiotics were fed for 3 days and observed by fluorescence microscope. Individuals with green fluorescence are infected individuals, and individuals without green fluorescence are non-infected individuals. According to the genome information of Bombyx mori, STS molecular markers were designed to map the sav gene. The sav gene was located in the third linkage group. The genetic distance of the genetic linkage map was 48.7 cm, and the SAV gene was located at 37.6cM. The distance between T0399 and T03147 markers and sav gene was 5.1 cm and 0.4 cm, respectively.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S882
本文编号：2494003
[Abstract]:With the development of social economy, air pollution is a big problem in many fields. Fluoride is an important part of polluted air, it is not only harmful to human body, but also pollutes trees and silkworm, and endangers sericulture industry. The results showed that the fluorine tolerance of Bombyx mori was mainly controlled by the dominant major gene (Dtf), and it had been mapped to the 12th linkage group of Bombyx mori by SSR markers. In our experiment, the parents of Pinus tabulaeformis were sensitive to fluorine, and the larvae died when fed with mulberry leaves soaked with 50 mg/kg NaF mulberry leaves, and when fed with mulberry leaves soaked with 100 mg/kg NaF, all the larvae died. However, individuals with resistance genes hardly had toxic symptoms when they were fed mulberry leaves soaked with 200 mg/kg NaF. Bombyx mori is the representative of Lepidoptera. The main feature of its wings is that it is covered with a layer of scaly hair. In the process of seed production, a large number of scales and hairs will fall off, drift into dust in the air, pollute the working environment, and endanger the health of seed production staff. Sericulture scholars found its mutant (adult wing without scaly hair) through resource investigation and collection. Through genetic analysis, it was found that the trait was controlled by recessive gene (nlw), and its fine mapping was carried out by molecular marker technique. In this experiment, Pinus elliottii was used as reincarnation parent and crossed with U13 and T6 varieties. First, the cyanine pine variety was used as the female parent, T6 strain as the male parent, then F1 generation as the female parent and U13 strain, and the progeny BC1 was backcrossed with the female parent of the juniper pine strain, and then the F1 generation was used as the female parent to cross with the U13 strain. In the next generation of BC2 generation, the male individuals with the best economic traits were selected as the male parent and the female parent of Pinus elliottii as backcross. In each backcross progenies, SSR,STS markers linked to fluorine-tolerant genes Dtf (S1214 and S121201) and molecular markers (S1305 and cash2p) linked to scalyless wing gene (nlw) were used to detect male moths. If any marker shows the homozygous type of cyanine pine, the offspring of the individual will be eliminated, leaving only the progenies of S1214 and S121201 loci as hetero-type of cyanine pine and T6 and the progenies of S1305 and cash2p loci showing cyanine pine and U13 heterozygous type. The detection method was used to backcross to BC6 generation. After that, the population obtained by self-cross fed mulberry leaves contaminated with 200 mg/kg NaF, and then survived, the silkworm, which had no scaly wings and good economic characters, was selected, and the next generation of BC_6F_3. was produced by self-cross. Molecular markers were used to select individuals who were homozygous at Dtf and nlw loci. In this way, silkworm (Bombyx mori) with good traits and high fluorine tolerance without scaly hair was produced. The nuclear polyhedrosis virus (Autographed California multiple nucleopolyhedrovirus), as a representative species of baculovirus, can infect many hosts. It was found that the recombinant AcMNPV could infect some Bombyx mori varieties by injection and vaccination, and the infection of AcMNPV to Bombyx mori was controlled by recessed single gene (susceptible to AcMNPV,sav. Bombyx mori non-infected variety C108 and infected variety P50 were used as parents to match positive and negative cross population, and backcross population (P50 脳 C108) 脳 P50 脳 (P50 脳 C108) was obtained, which was recorded as BC1F and BC1M.. The fifth instar larvae of Bombyx mori were injected with recombinant AcMNPV virus containing GFP markers during the feeding process, and the mulberry leaves soaked with antibiotics were fed for 3 days and observed by fluorescence microscope. Individuals with green fluorescence are infected individuals, and individuals without green fluorescence are non-infected individuals. According to the genome information of Bombyx mori, STS molecular markers were designed to map the sav gene. The sav gene was located in the third linkage group. The genetic distance of the genetic linkage map was 48.7 cm, and the SAV gene was located at 37.6cM. The distance between T0399 and T03147 markers and sav gene was 5.1 cm and 0.4 cm, respectively.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S882
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