利用MAS技術選育耐氟且無鱗毛家蠶新品種及AcMNPV敏感性基因的定位分析
發(fā)布時間：2019-06-06 02:30
【摘要】：隨著社會經濟的發(fā)展,空氣污染在許多領域都是一個大問題。氟化物是污染空氣的重要組成部分,它不僅對人體有害,還污染樹木和桑蠶,危害著蠶桑產業(yè)。經研究表明家蠶耐氟性主要由顯性主效基因(Dtf)控制著,并已利用SSR標記將其定位于家蠶第12連鎖群。在我們的試驗中菁松親本是對氟敏感的,當喂食浸漬50 mg/kg NaF的桑葉時就會產生中毒癥狀,當喂食浸漬100 mg/kg NaF的桑葉時,幼蟲全部死亡。而帶有抗性基因的個體喂食浸漬200 mg/kg NaF的桑葉時幾乎不產生中毒癥狀。家蠶是鱗翅目的代表,它的翅膀的主要特征是上面覆蓋有一層鱗毛。在制種過程中會有大量的鱗毛脫落,飄散在空氣中而成微塵,污染工作環(huán)境,危害制種工作人員的健康。蠶業(yè)學者通過資源考察與采集發(fā)現了它的突變體(成蟲翅無鱗毛),經遺傳分析,發(fā)現該性狀是由隱性基因(nlw)控制的,并通過分子標記技術將其精細定位。本實驗以菁松為輪回親本,先后與U13和T6品種雜交。首先以菁松品種為母本,T6品系為父本雜交,然后以F1代為母本與U13品系雜交,后代BC1再與菁松母本回交,產生的下一代的BC2代中篩選經濟性狀最好的雄性個體作為父本與菁松母本回交。在每一次回交后代中,都用與家蠶耐氟性基因Dtf連鎖的SSR、STS標記(S1214和S121201)以及與無鱗毛翅基因(nlw)連鎖的分子標記(S1305和cash2p)來檢測雄蛾子基因型。如果任一標記表現為菁松的純合型,則該個體的后代予以淘汰,只留S1214和S121201位點表現為菁松與T6的雜合型并且在S1305和cash2p位點表現為菁松和U13雜合型的個體的后代,采用這種檢測方法連續(xù)回交至BC6代。之后通過自交得到的群體喂食被200 mg/kg NaF污染的桑葉,然后存活下來個體選擇無鱗毛翅且經濟性狀良好的家蠶被選擇出來,自交產生下一代BC_6F_3。采用分子標記選出那些在Dtf和nlw位點純合的個體。這樣,具有良好性狀,高耐氟性并無鱗毛的家蠶產生了。苜蓿銀紋夜蛾核型多角體病毒(Autographed California multiple nucleopolyhedrovirus)作為桿狀病毒的代表種,能夠感染許多宿主。研究發(fā)現,通過注射接種的方法,重組AcMNPV可以感染部分家蠶品種,且家蠶對AcMNPV的感染性由隱性單基因(susceptible to AcMNPV,sav)控制。以家蠶不感染品種C108和感染品種P50為親本組配正反交群體,并獲得回交群體(P50×C108)×P50和P50×(P50×C108),記作BC1F和BC1M。在家蠶飼養(yǎng)過程中將分離群體的五齡幼蟲注射含有GFP標記的重組AcMNPV病毒,并喂養(yǎng)浸漬有抗生素的桑葉3天后用熒光顯微鏡區(qū)觀察。有綠色熒光的個體為感染個體,沒有綠色熒光的個體為不感染個體。根據家蠶基因組信息,設計STS分子標記對sav基因進行定位分析,將sav基因定位在第3連鎖群,繪制的遺傳連鎖圖的遺傳距離為48.7cM,sav基因位于37.6cM處,T0399和T03147標記與sav基因的距離最近,分別是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.
【學位授予單位】：江蘇科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S882
本文編號：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.
【學位授予單位】：江蘇科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S882
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