大豆胚尖再生体系的优化及抗逆基因GmPK的遗传转化研究
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摘要
干旱和土壤盐渍化是一个严重的全球性问题,在我国干旱面积、盐碱地和次生盐碱化耕地的面积逐年增加,制约着我国农作物的生产。因此,对盐碱地的治理、开发及抗逆新品种的培育成为近年来的研究热点和重点。大豆是世界上重要的粮食和油料作物,是人类植物蛋白的主要来源,但受环境等影响,大豆的产量和品质受到一定制约。利用农杆菌介导法将抗逆基因转入大豆,培育大豆抗逆新品种,是目前遗传转化研究的重点。本研究以河北省8个优质大豆品种为试验材料(冀豆7号、冀豆17号、五星2号、nf58、冀黄13号、五星1号、五星3号和中黄13号),通过形态学方法初步确定了其盐敏感性。利用其中4个大豆品种(五星1号、五星2号、五星3号、nf58)和高蛋白品种冀豆12号为材料,优化了大豆胚尖再生体系,与体细胞胚发生途径相比再生效率明显提高。在此基础上,以五星2号胚尖外植体作为受体系统,利用农杆菌介导法进行了抗逆基因GmPK(SnRK2亚家族的大豆蛋白激酶基因)的遗传转化。结果表明:
1、在NaCl浓度为35 mmol/L时,五星2号、五星3号和冀豆17号三个基因型的株高和侧根长度受到盐胁迫的抑制,与对照差异显著。当NaCl浓度达70 mmol/L时,8个供试基因型的株高、下胚轴长度和侧根长度均受到一定程度的抑制。
2、不同基因型以及培养基中的激素浓度影响胚尖再生。5个供试品种中,冀豆12号的再生体系表现最佳。诱导培养基中附加低浓度的6-BA(≤0.5 mg/L),胚尖外植体的发芽率较高,平均丛生芽数较多。茎伸长培养基中附加0.5 mg/L GA3的茎伸长效果优于附加0.2 mg/L的6-BA和IBA。利用1/2 MS基本培养基附加蔗糖20 g/L和IBA 0.5 mg/L的生根培养基,诱导生根的效果最好,生根率达到85.4%。
3、以五星2号为试验材料,利用农杆菌介导法进行GmPK基因的遗传转化。通过辅助超声波处理、共培养阶段附加2 mg/L的AgNO3和2 mg/L脯氨酸明显提高了胚尖转化的PCR阳性率(胚尖转化的侵染率);炼苗移栽成活的再生植株又经PCR及PCR-Southern blotting验证,初步证明GmPK基因已经整合至大豆基因组中,转化率为0.6%。
Drought and soil salinization is a serious global problem. In China, the drought area, saline, and secondary salinization of cultivated land increases every year, restricting the production of crops in China. Therefore, saline control, exploitation and stress resistance of new varieties has become a research hotspot in recent years. Soybean is the world's major grain and oil crops, the main human source of vegetable protein, but by the environmental impact, the soybean's output and quality is restricted. Agrobacterium-mediated method with the resistance gene into soybean, cultivation of new stress resistance varieties of soybean, is the focus of genetic transformation study. In this study, eight high-quality soybean varieties in Hebei Province as the test material are initially identified its salt sensitivity by morphology(jidou 7,jidou 17,wuxing 1,wuxing 2, wuxing 3,nf 58, jihuang 13, zhonghuang 13). Using four soybean varieties (wuxing 1, wuxing 2, wuxing 3, nf 58) and high protein varieties Jidou 12 as materials, the soybean embryonic tip regeneration system are optimizated, efficiency is improved significantly comparing with the somatic embryos system. On this basis, the wuxing 2 embryo tip regeneration system as the receptor system, genetic transformation of resistance gene GmPK (soybean protein kinase gene of SnRK2 family) is undertaken using agrobacterium-mediated method. The results show that:
1、Under NaCl concentration of 35 mmol/L, the height and length of root of jidou 17, wuxing 2 and wuxing 3 were inhibited, and the difference is significant compared with the control. Under NaCl concentration of 70 mmol/L, height, length of root and length of hypocotyl of 8 genotypes tested were significantly inhibited.
2、Different genotypes as well as the concentration of hormone in media impact on embryonic tip regeneration. In the five varieties, performance of Jidou 12 regeneration system is the best. Tip explants germination rate and the average amount of multiple shoots are higher on the induction medium supplemented with 0.5 mg/L 6-BA or with 0.2 mg/L IBA. Stem elongation medium supplemented with 0.5 mg / L GA3 was better than that with 0.2 mg/L 6-BA and IBA. The best effect of rooting was from one using 1/2 MS basic medium with addition of 20 g/L sugar and 0.5 mg/L IBA, rooting rate is 85.4%.
3、The wuxing 2 as experimental material, genetic transformation of GmPK is undertaken using Agrobacterium-mediated method. Through the auxiliary ultrasonic treatment and addition of 2 mg/L of AgNO3 and 2 mg/L of Pro in co-culture period, PCR-positive rate of embryo tip conversion is significantly improved (the infection rate of embryo tip conversion); survival of transplanted regeneration is confirmed by PCR and PCR-Southern, it proves that gene GmPK has been integrated into the genome of soybean, conversion rate was 0.6%.
1、在NaCl浓度为35 mmol/L时,五星2号、五星3号和冀豆17号三个基因型的株高和侧根长度受到盐胁迫的抑制,与对照差异显著。当NaCl浓度达70 mmol/L时,8个供试基因型的株高、下胚轴长度和侧根长度均受到一定程度的抑制。
2、不同基因型以及培养基中的激素浓度影响胚尖再生。5个供试品种中,冀豆12号的再生体系表现最佳。诱导培养基中附加低浓度的6-BA(≤0.5 mg/L),胚尖外植体的发芽率较高,平均丛生芽数较多。茎伸长培养基中附加0.5 mg/L GA3的茎伸长效果优于附加0.2 mg/L的6-BA和IBA。利用1/2 MS基本培养基附加蔗糖20 g/L和IBA 0.5 mg/L的生根培养基,诱导生根的效果最好,生根率达到85.4%。
3、以五星2号为试验材料,利用农杆菌介导法进行GmPK基因的遗传转化。通过辅助超声波处理、共培养阶段附加2 mg/L的AgNO3和2 mg/L脯氨酸明显提高了胚尖转化的PCR阳性率(胚尖转化的侵染率);炼苗移栽成活的再生植株又经PCR及PCR-Southern blotting验证,初步证明GmPK基因已经整合至大豆基因组中,转化率为0.6%。
Drought and soil salinization is a serious global problem. In China, the drought area, saline, and secondary salinization of cultivated land increases every year, restricting the production of crops in China. Therefore, saline control, exploitation and stress resistance of new varieties has become a research hotspot in recent years. Soybean is the world's major grain and oil crops, the main human source of vegetable protein, but by the environmental impact, the soybean's output and quality is restricted. Agrobacterium-mediated method with the resistance gene into soybean, cultivation of new stress resistance varieties of soybean, is the focus of genetic transformation study. In this study, eight high-quality soybean varieties in Hebei Province as the test material are initially identified its salt sensitivity by morphology(jidou 7,jidou 17,wuxing 1,wuxing 2, wuxing 3,nf 58, jihuang 13, zhonghuang 13). Using four soybean varieties (wuxing 1, wuxing 2, wuxing 3, nf 58) and high protein varieties Jidou 12 as materials, the soybean embryonic tip regeneration system are optimizated, efficiency is improved significantly comparing with the somatic embryos system. On this basis, the wuxing 2 embryo tip regeneration system as the receptor system, genetic transformation of resistance gene GmPK (soybean protein kinase gene of SnRK2 family) is undertaken using agrobacterium-mediated method. The results show that:
1、Under NaCl concentration of 35 mmol/L, the height and length of root of jidou 17, wuxing 2 and wuxing 3 were inhibited, and the difference is significant compared with the control. Under NaCl concentration of 70 mmol/L, height, length of root and length of hypocotyl of 8 genotypes tested were significantly inhibited.
2、Different genotypes as well as the concentration of hormone in media impact on embryonic tip regeneration. In the five varieties, performance of Jidou 12 regeneration system is the best. Tip explants germination rate and the average amount of multiple shoots are higher on the induction medium supplemented with 0.5 mg/L 6-BA or with 0.2 mg/L IBA. Stem elongation medium supplemented with 0.5 mg / L GA3 was better than that with 0.2 mg/L 6-BA and IBA. The best effect of rooting was from one using 1/2 MS basic medium with addition of 20 g/L sugar and 0.5 mg/L IBA, rooting rate is 85.4%.
3、The wuxing 2 as experimental material, genetic transformation of GmPK is undertaken using Agrobacterium-mediated method. Through the auxiliary ultrasonic treatment and addition of 2 mg/L of AgNO3 and 2 mg/L of Pro in co-culture period, PCR-positive rate of embryo tip conversion is significantly improved (the infection rate of embryo tip conversion); survival of transplanted regeneration is confirmed by PCR and PCR-Southern, it proves that gene GmPK has been integrated into the genome of soybean, conversion rate was 0.6%.
引文
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