轉(zhuǎn)Bt雙價基因甘藍(lán)的抗蟲性及遺傳穩(wěn)定性研究
發(fā)布時間：2019-05-17 16:33
【摘要】：結(jié)球甘藍(lán)簡稱甘藍(lán),是十字花科蕓薹屬的一種重要蔬菜。近年來,甘藍(lán)上的蟲害日趨嚴(yán)重,尤其是鱗翅目的小菜蛾和菜青蟲等危害最為嚴(yán)重。培育抗蟲品種是防治害蟲最理想的方法,但在甘藍(lán)類蔬菜中缺乏抗蟲種質(zhì)資源,采用基因工程技術(shù)將外源抗蟲基因轉(zhuǎn)入甘藍(lán),可為育種提供有效的抗蟲材料。 Bt crylIa8和cry1Ba3均是具有自主知識產(chǎn)權(quán)的新型殺蟲蛋白基因,其編碼蛋白對小菜蛾等鱗翅目害蟲具有高毒力,且Cry1Ia8. Cry1Ba3與生產(chǎn)上常用的Cry1A類蛋白之間沒有交互抗性。利用cry1Ia8和cry1Ba3進行轉(zhuǎn)雙價基因甘藍(lán)的研制,不但可以提高甘藍(lán)的抗蟲性,還可以降低害蟲快速產(chǎn)生抗性的風(fēng)險。此外,在獲得轉(zhuǎn)抗蟲基因甘藍(lán)材料后,研究轉(zhuǎn)基因甘藍(lán)的遺傳穩(wěn)定性、抗蟲性和安全性,可為甘藍(lán)抗蟲育種提供理論指導(dǎo),加速轉(zhuǎn)基因甘藍(lán)在實踐中的應(yīng)用。 本研究采用農(nóng)桿菌介導(dǎo)法將cry1Ia8和cry1Ba3基因同時導(dǎo)入甘藍(lán)高代自交系,然后對轉(zhuǎn)基因甘藍(lán)的遺傳穩(wěn)定性、抗蟲性和安全性等方面進行了研究,主要結(jié)果如下： 1.轉(zhuǎn)cry1Ia8-cry1Ba3雙價基因甘藍(lán)純系的獲得 以重組質(zhì)粒表達載體pCSIaBaN(含有cry1Ia8和cry1Ba3)轉(zhuǎn)化甘藍(lán),共獲得53株卡那霉素抗性植株,其中的33株在RNA和蛋白質(zhì)水平均得到了表達；轉(zhuǎn)雙價基因植株在離體條件下對敏感小菜蛾和CrylAc抗性小菜蛾均具有很強的抗性；通過連續(xù)多代自交和篩選獲得9個轉(zhuǎn)基因甘藍(lán)純系,并利用其配制了雜交組合。 2.轉(zhuǎn)crylIa8-cry1Ba3雙價基因甘藍(lán)的遺傳穩(wěn)定性分析 在獲得T0、T1、T2、T3四個世代轉(zhuǎn)cry1Ia8-crylBa3雙價基因植株的基礎(chǔ)上,對外源基因的遺傳穩(wěn)定性進行了研究：T0-1、T0-9、T0-13、T0-17等4個單拷貝轉(zhuǎn)化植株外源目的基因的分離規(guī)律均符合單基因顯性遺傳；Cry1Ia8、Cry1Ba3蛋白含量在同一轉(zhuǎn)化事件的不同世代間均沒有顯著性差異；外源基因的導(dǎo)入未對轉(zhuǎn)基因甘藍(lán)純合株系及其雜交后代的株高、開展度、外葉數(shù)、球高、球?qū)挕⒅行闹L和單球重等主要農(nóng)藝性狀產(chǎn)生影響。 3.轉(zhuǎn)基因甘藍(lán)的抗蟲性評價 通過網(wǎng)室和田間抗蟲性的鑒定、田間主要害蟲種類與數(shù)量的調(diào)查、田間主要害蟲離體生物活性的測定,表明轉(zhuǎn)雙價基因甘藍(lán)對小菜蛾和菜青蟲具有極強的抗性,而對甘藍(lán)夜蛾和甜菜夜蛾沒有明顯抗性。 4.轉(zhuǎn)基因甘藍(lán)花粉和高濃度的Bt蛋白對蜜蜂幼蟲存活率的影響 將轉(zhuǎn)cry1Ia8-crylBa3雙價基因甘藍(lán)花粉、Cry1Ia8蛋白(3000ng/ml)及Cry1Ba3蛋白(3000ng/ml)直接飼喂意大利蜜蜂的工蜂幼蟲,20天后,與取食對照甘藍(lán)花粉、雜花粉相比,取食轉(zhuǎn)基因甘藍(lán)花粉和Bt蛋白的蜜蜂存活率均未受到負(fù)面影響。
[Abstract]:Cabbage, abbreviated as cabbage, is an important vegetable of the genus Cruciferae. In recent years, the pests on cabbage have become more and more serious, especially the Plutella xylostella and cabbage insects in Lepidoptera. Breeding insect-resistant varieties is the most ideal method to control pests, but there is a lack of insect-resistant germplasm resources in cabbage vegetables. Genetic engineering technology can be used to transfer exogenous insect-resistant genes into cabbage, which can provide effective insect-resistant materials for breeding. Both Bt crylIa8 and cry1Ba3 are novel insecticidal protein genes with independent intellectual property rights. Their encoded proteins are highly virulent to Lepidoptera pests such as Plutella xylostella, and Cry1Ia8. is highly toxic to Lepidoptera pests such as Plutella xylostella. There is no cross resistance between Cry1Ba3 and Cry1A proteins commonly used in production. The preparation of transgenic cabbage with bivalent gene by cry1Ia8 and cry1Ba3 can not only improve the insect resistance of cabbage, but also reduce the risk of rapid resistance of pests. In addition, after obtaining transgenic cabbage materials, the genetic stability, insect resistance and safety of transgenic cabbage can be studied, which can provide theoretical guidance for insect-resistant breeding of cabbage and accelerate the application of transgenic cabbage in practice. In this study, cry1Ia8 and cry1Ba3 genes were introduced into cabbage inbred lines at the same time by Agrobacterium tumefaciens, and then the genetic stability, insect resistance and safety of transgenic cabbage were studied. The main results are as follows: 1. Transgenic cabbage lines with cry1Ia8-cry1Ba3 bivalent gene were transformed into cabbage with recombinant plasmid expression vector pCSIaBaN (containing cry1Ia8 and cry1Ba3). A total of 53 kanamycin resistant plants were obtained, 33 of which were expressed at RNA and protein levels. The transgenic plants were highly resistant to sensitive Plutella xylostella and CrylAc resistant Plutella xylostella in vitro, and 9 transgenic cabbage lines were obtained by continuous inbreeding and screening, and the hybrid combinations were prepared by using them. 2. The genetic stability of transgenic cabbage with crylIa8-cry1Ba3 bivalent gene was studied on the basis of obtaining four generations of transgenic cry1Ia8-crylBa3 bivalent gene plants: T0, T1, T2, T3. The genetic stability of foreign genes was studied. The isolation of foreign target genes from four single copy transformed plants, such as T0 13 and T0 17, were in accordance with the dominant inheritance of single gene. There was no significant difference in Cry1Ia8,Cry1Ba3 protein content among different generations of the same transformation event. The introduction of foreign genes had no effect on the plant height, development degree, number of outer leaves, ball height, ball width, central column length and single bulb weight of transgenic cabbage homozygous lines and their hybrid progenies. 3. The evaluation of insect resistance of transgenic cabbage was carried out through the identification of insect resistance in net room and field, the investigation of the species and quantity of main pests in the field, and the determination of in vitro biological activity of the main pests in the field. The results showed that transgenic cabbage had strong resistance to Plutella xylostella and Plutella xylostella, but no obvious resistance to Plutella xylostella and Plutella xylostella. 4. Effects of transgenic cabbage pollen and high concentration of Bt protein on the survival rate of honeybee larvae were directly fed with cry1Ia8-crylBa3 bivalent cabbage pollen, Cry1Ia8 protein (3000ng/ml) and Cry1Ba3 protein (3000ng/ml). After 20 days, compared with the control cabbage pollen and miscellaneous pollen, the survival rate of bees fed on transgenic cabbage pollen and Bt protein was not negatively affected.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：S635.1
本文編號：2479245
[Abstract]:Cabbage, abbreviated as cabbage, is an important vegetable of the genus Cruciferae. In recent years, the pests on cabbage have become more and more serious, especially the Plutella xylostella and cabbage insects in Lepidoptera. Breeding insect-resistant varieties is the most ideal method to control pests, but there is a lack of insect-resistant germplasm resources in cabbage vegetables. Genetic engineering technology can be used to transfer exogenous insect-resistant genes into cabbage, which can provide effective insect-resistant materials for breeding. Both Bt crylIa8 and cry1Ba3 are novel insecticidal protein genes with independent intellectual property rights. Their encoded proteins are highly virulent to Lepidoptera pests such as Plutella xylostella, and Cry1Ia8. is highly toxic to Lepidoptera pests such as Plutella xylostella. There is no cross resistance between Cry1Ba3 and Cry1A proteins commonly used in production. The preparation of transgenic cabbage with bivalent gene by cry1Ia8 and cry1Ba3 can not only improve the insect resistance of cabbage, but also reduce the risk of rapid resistance of pests. In addition, after obtaining transgenic cabbage materials, the genetic stability, insect resistance and safety of transgenic cabbage can be studied, which can provide theoretical guidance for insect-resistant breeding of cabbage and accelerate the application of transgenic cabbage in practice. In this study, cry1Ia8 and cry1Ba3 genes were introduced into cabbage inbred lines at the same time by Agrobacterium tumefaciens, and then the genetic stability, insect resistance and safety of transgenic cabbage were studied. The main results are as follows: 1. Transgenic cabbage lines with cry1Ia8-cry1Ba3 bivalent gene were transformed into cabbage with recombinant plasmid expression vector pCSIaBaN (containing cry1Ia8 and cry1Ba3). A total of 53 kanamycin resistant plants were obtained, 33 of which were expressed at RNA and protein levels. The transgenic plants were highly resistant to sensitive Plutella xylostella and CrylAc resistant Plutella xylostella in vitro, and 9 transgenic cabbage lines were obtained by continuous inbreeding and screening, and the hybrid combinations were prepared by using them. 2. The genetic stability of transgenic cabbage with crylIa8-cry1Ba3 bivalent gene was studied on the basis of obtaining four generations of transgenic cry1Ia8-crylBa3 bivalent gene plants: T0, T1, T2, T3. The genetic stability of foreign genes was studied. The isolation of foreign target genes from four single copy transformed plants, such as T0 13 and T0 17, were in accordance with the dominant inheritance of single gene. There was no significant difference in Cry1Ia8,Cry1Ba3 protein content among different generations of the same transformation event. The introduction of foreign genes had no effect on the plant height, development degree, number of outer leaves, ball height, ball width, central column length and single bulb weight of transgenic cabbage homozygous lines and their hybrid progenies. 3. The evaluation of insect resistance of transgenic cabbage was carried out through the identification of insect resistance in net room and field, the investigation of the species and quantity of main pests in the field, and the determination of in vitro biological activity of the main pests in the field. The results showed that transgenic cabbage had strong resistance to Plutella xylostella and Plutella xylostella, but no obvious resistance to Plutella xylostella and Plutella xylostella. 4. Effects of transgenic cabbage pollen and high concentration of Bt protein on the survival rate of honeybee larvae were directly fed with cry1Ia8-crylBa3 bivalent cabbage pollen, Cry1Ia8 protein (3000ng/ml) and Cry1Ba3 protein (3000ng/ml). After 20 days, compared with the control cabbage pollen and miscellaneous pollen, the survival rate of bees fed on transgenic cabbage pollen and Bt protein was not negatively affected.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：S635.1
【參考文獻】
相關(guān)期刊論文 前10條
1 唐克軒,徐亞男,李旭峰,孫小芬;表達雪花蓮?fù)庠茨鼗虻挠筒宿D(zhuǎn)基因植株的獲得(英文)[J];復(fù)旦學(xué)報(自然科學(xué)版);2001年06期
2 閻文昭,吳潔,王大一,譚文芳;將水稻半胱氨酸蛋白酶抑制劑(Oryzacystatin I)基因?qū)敫适砥贩N[J];分子植物育種;2004年02期
3 周靜;弭曉菊;崔繼哲;;植物轉(zhuǎn)基因的非孟德爾遺傳[J];分子植物育種;2006年05期
4 祝長青;趙惠新;李艷紅;覃建兵;;轉(zhuǎn)基因小麥后代外源目的基因的遺傳分析[J];干旱區(qū)研究;2008年06期
5 胡桂兵;張上隆;徐昌杰;林順權(quán);;柑橘密碼子用法分析[J];果樹學(xué)報;2006年03期
6 毛慧珠,唐惕,曹湘玲,白永延,郭培福,符文俊;抗蟲轉(zhuǎn)基因甘藍(lán)及其后代的研究[J];中國科學(xué)C輯:生命科學(xué);1996年04期
7 佘建明,蔡小寧,朱衛(wèi)民,張初賢,丁萬霞,李建斌,李彬,朱禎;結(jié)球甘藍(lán)抗蟲轉(zhuǎn)基因植株及其后代的抗性表現(xiàn)[J];江蘇農(nóng)業(yè)學(xué)報;2001年02期
8 佘建明,蔡小寧,朱禎,徐鶴林,朱衛(wèi)民,吳敬音,王愛民,丁萬霞;結(jié)球甘藍(lán)的離體再生及基因轉(zhuǎn)化條件研究[J];江蘇農(nóng)業(yè)學(xué)報;1996年03期
9 郭芳;梁革梅;曹廣春;高希武;郭予元;;棉鈴蟲對Bt殺蟲劑的抗性遺傳方式[J];昆蟲學(xué)報;2010年04期
10 王志斌,郭三堆;表達cryIA/gna雙價抗蟲基因煙草兼抗棉鈴蟲和蚜蟲[J];科學(xué)通報;1999年19期
相關(guān)博士學(xué)位論文 前1條
1 楊宙;雙價Bt抗蟲水稻的培育和轉(zhuǎn)基因水稻回交效應(yīng)分析[D];華中農(nóng)業(yè)大學(xué);2011年
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