甘蔗C_4磷酸烯醇式丙酮酸羧化酶基因植物表达载体构建及转化大豆研究
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摘要
本研究将来自甘蔗含全部内含子、部分5’侧端利3’末端、全长6.8Kb的C_4磷酸烯醇式丙酮酸羧化酶基因(甘蔗C_4Ppc)用不同启动强度的启动子插入到植物表达载体pBI121和pCAMBIA1301中，获得植物表达载体pLApepc、pBIpepc和pCBIpepc，然后导入农杆菌LBA4404，经适宜的助介方法转化大豆子叶节，在适宜的选择压下获得pBIpepc大豆转化植株；用花粉管直接导入法获得pBIpepc大豆转基因植株；通过PCR和斑点Southern杂交鉴定甘蔗C_4磷酸烯醇式丙酮酸羧化酶基因的转化整合。同时，为了比较不同类型启动子对甘蔗C_4Ppc转入C_3双子叶植物的影响，用农杆菌介导法转化烟草获得木材植物表达载体pLApepc、pBIpepc和pCBIpepc烟草转化植株，通过PCR和斑点Southern杂交及GUS检测鉴定甘蔗C_4磷酸烯醇式丙酮酸羧化酶基因的转化整合；不同类型启动子对甘蔗C_4Ppcc转入烟草的表达检测正在进行之中。
研究工作获得如下几个方面的结果：
1、根据两端测序利用序列同源性比较确定了用于转化的甘蔗C_4Ppc基因5’侧端与3’末端。
2、根据用于转化的甘蔗C_4Ppc基因有全部内含子、部分5’侧端、3’末端的特点，分别构建了无启动子、CaMV35S启动子和CaMV2x35S启动子的植物表达载体pLApepc、PBIpepc和pCBIpepc。其中，pBIpepc抗性选择标记基因为nptⅡ，pLApepc和pCBIpepc抗性选择标记基因为hyg。
3、研究了影响农杆菌介导转化主要影响因素，受体材料的生理状态及表达载体是农杆菌介导转化中的关键因素。
4、建立了大豆子叶节高效再生系统及有效而又较简捷的大豆子叶节农杆菌介导转化再生系统。成熟的大豆种子经选种消毒处理播种于透气性良好的MSB基上，并经适宜的激素与温度处理获得的大豆子叶节成为农杆菌介导转化的良好受体，采用适宜的子叶节切取方式、AS浓度、共培养时间与浸染时间，合理的助介处理方法等有利于提高大豆转化抗性苗再生频率。
5、获得了甘蔗C_4Ppc基因表达载体pBIpepc大豆转化植株，经PCR检测及斑点Sourthern杂交检测，证明C_4Ppc基因已整合进大豆基因组中。
6、获得了甘蔗C_4Ppc基因表达载体pBIpepc烟草植株，经PCR检测及斑点Sourthern杂交检测，证明C_4Ppc基因已整合进烟草基因组中。
7、获得了甘蔗C_4Ppc基因表达载体pLApepc、pCBIpepc烟草转化植株，经GUS检测初步证明外源基因已整合进烟草基因组中。
8、观察到部分烟草、大豆转化植株的生长较非转化植株生长慢。
Three plant expression vectors were constructed by inserting the C4 phosphoenolpyruvate carboxylase gene which includes all introns; partial 5'flanking and partial 3'ending sequence with 6.8 Kb from sugarcane, into pBI121 getting pBIpepc with a one copy CaMV35S as promoter and nptll as selectable marker gene; into pCAMBIA1301 getting pLApepc with no promoter and hyg as selectable marker gene , into pCAMBIA1301 getting pCBIpepc with a two copy CaMV35S as promoter and hyg as selectable marker gene. Then, they were introduced into Agrobacterium tumefaciens strain LBA4404 via the freeze-melting. The protocol of transformation for soybean(Glycine max (L.) Merrill) cotyledonary node has been optimized. Soybean's cotyledonary node which was cultured with 1 .Omg/L BA and low temperature for a few days was transformed by Sonication-Assisted Agrobacterium tumefaciens-mediated method. The transformed cotyledonary node and shoots were screened under the selective pressure of Kan or Hyg. The transformed plants of pBIpepc from Agrobacterium tumefaciens-mediated and pollen-tube were detected by PCR and dot Southern bolting. In order to compare the expression efficiency of different types of promoters for sugarcane C4 phosphoenolpyruvate carboxylase gene in transgenic €3 plants, Three plant expression vectors were transformed into tobacco by Agrobacterium tumefaciens . The results were as follows:
1.The direction of sugarcane C4 phosphoenolpyruvate carboxylase gene was identified by partial 5'flanking and 3'ending sequence through the Blast n analysis.
2.Three plant expression vectors pLApepc, pBIpepc and pCBIpepc were constructed by inserted the C4 phosphoenolpyruvate carboxylase gene from sugarcane into pBI121 and pCAMBIA 1301. The selectable marker gene of pBIpepc was nptll and the selectable marker gene of pLApepc and pCBIpepc was hyg.
3.The main factors of transformation mediated by Agrobacterium tumefaciens were investigated and the physiological character of explant and plant expression vector were very important for
transformation mediated by Agrobacterium tumefaciens.
4.An efficient protocol for plant regeneration from soybean cotyledonary node and for soybean
cotyledonary node transformation mediated by Agrobacterium tumefaciens has been established.
Some factors may be important for the success:(l) cotyledonary node were induced as
competent explants such as 6-BA and culturing in low temperature after seed germination in
aerated MSB media , (2) Several transformation conditions were combined to improve the
activation of vir genes and T-DNA transfer, such as methods of incising soybean cotyledonary
node; Acetosyringone concentrations; co-culture time; infection time and acid infection
condition and low co- culture temperature.
S.Transgenic soybean plants were recovered from co-cultrue of cotyledonary node with
Agrobacterium tumefaciens I pBIpepc and transformants were obtained after long Kan resistance
selection, 8 of them have been detected by PCR and dot Southern bloting, and the DNA of 2
plants showed positive.
6. Tobacco transformants were obtained for pLApepc; pBIpepc; pCBIpepc and transformants
have been detected by GUS.The pBIpepc transformants have been detected by PCR and dot
Southern bloting.
7.The slow growth in a few transformants of soybean and tobacco has been observed .
研究工作获得如下几个方面的结果：
1、根据两端测序利用序列同源性比较确定了用于转化的甘蔗C_4Ppc基因5’侧端与3’末端。
2、根据用于转化的甘蔗C_4Ppc基因有全部内含子、部分5’侧端、3’末端的特点，分别构建了无启动子、CaMV35S启动子和CaMV2x35S启动子的植物表达载体pLApepc、PBIpepc和pCBIpepc。其中，pBIpepc抗性选择标记基因为nptⅡ，pLApepc和pCBIpepc抗性选择标记基因为hyg。
3、研究了影响农杆菌介导转化主要影响因素，受体材料的生理状态及表达载体是农杆菌介导转化中的关键因素。
4、建立了大豆子叶节高效再生系统及有效而又较简捷的大豆子叶节农杆菌介导转化再生系统。成熟的大豆种子经选种消毒处理播种于透气性良好的MSB基上，并经适宜的激素与温度处理获得的大豆子叶节成为农杆菌介导转化的良好受体，采用适宜的子叶节切取方式、AS浓度、共培养时间与浸染时间，合理的助介处理方法等有利于提高大豆转化抗性苗再生频率。
5、获得了甘蔗C_4Ppc基因表达载体pBIpepc大豆转化植株，经PCR检测及斑点Sourthern杂交检测，证明C_4Ppc基因已整合进大豆基因组中。
6、获得了甘蔗C_4Ppc基因表达载体pBIpepc烟草植株，经PCR检测及斑点Sourthern杂交检测，证明C_4Ppc基因已整合进烟草基因组中。
7、获得了甘蔗C_4Ppc基因表达载体pLApepc、pCBIpepc烟草转化植株，经GUS检测初步证明外源基因已整合进烟草基因组中。
8、观察到部分烟草、大豆转化植株的生长较非转化植株生长慢。
Three plant expression vectors were constructed by inserting the C4 phosphoenolpyruvate carboxylase gene which includes all introns; partial 5'flanking and partial 3'ending sequence with 6.8 Kb from sugarcane, into pBI121 getting pBIpepc with a one copy CaMV35S as promoter and nptll as selectable marker gene; into pCAMBIA1301 getting pLApepc with no promoter and hyg as selectable marker gene , into pCAMBIA1301 getting pCBIpepc with a two copy CaMV35S as promoter and hyg as selectable marker gene. Then, they were introduced into Agrobacterium tumefaciens strain LBA4404 via the freeze-melting. The protocol of transformation for soybean(Glycine max (L.) Merrill) cotyledonary node has been optimized. Soybean's cotyledonary node which was cultured with 1 .Omg/L BA and low temperature for a few days was transformed by Sonication-Assisted Agrobacterium tumefaciens-mediated method. The transformed cotyledonary node and shoots were screened under the selective pressure of Kan or Hyg. The transformed plants of pBIpepc from Agrobacterium tumefaciens-mediated and pollen-tube were detected by PCR and dot Southern bolting. In order to compare the expression efficiency of different types of promoters for sugarcane C4 phosphoenolpyruvate carboxylase gene in transgenic €3 plants, Three plant expression vectors were transformed into tobacco by Agrobacterium tumefaciens . The results were as follows:
1.The direction of sugarcane C4 phosphoenolpyruvate carboxylase gene was identified by partial 5'flanking and 3'ending sequence through the Blast n analysis.
2.Three plant expression vectors pLApepc, pBIpepc and pCBIpepc were constructed by inserted the C4 phosphoenolpyruvate carboxylase gene from sugarcane into pBI121 and pCAMBIA 1301. The selectable marker gene of pBIpepc was nptll and the selectable marker gene of pLApepc and pCBIpepc was hyg.
3.The main factors of transformation mediated by Agrobacterium tumefaciens were investigated and the physiological character of explant and plant expression vector were very important for
transformation mediated by Agrobacterium tumefaciens.
4.An efficient protocol for plant regeneration from soybean cotyledonary node and for soybean
cotyledonary node transformation mediated by Agrobacterium tumefaciens has been established.
Some factors may be important for the success:(l) cotyledonary node were induced as
competent explants such as 6-BA and culturing in low temperature after seed germination in
aerated MSB media , (2) Several transformation conditions were combined to improve the
activation of vir genes and T-DNA transfer, such as methods of incising soybean cotyledonary
node; Acetosyringone concentrations; co-culture time; infection time and acid infection
condition and low co- culture temperature.
S.Transgenic soybean plants were recovered from co-cultrue of cotyledonary node with
Agrobacterium tumefaciens I pBIpepc and transformants were obtained after long Kan resistance
selection, 8 of them have been detected by PCR and dot Southern bloting, and the DNA of 2
plants showed positive.
6. Tobacco transformants were obtained for pLApepc; pBIpepc; pCBIpepc and transformants
have been detected by GUS.The pBIpepc transformants have been detected by PCR and dot
Southern bloting.
7.The slow growth in a few transformants of soybean and tobacco has been observed .
引文
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