cecropin B分泌型融合基因的构建及其转化柑桔的研究
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摘要
柑桔是世界最重要的商品水果之一,在所有水果种类中,其种植面积和产量均居第一位,是第五大国际贸易农产品。在全球138个国家和地区栽培柑桔,是我国南方农村的经济支柱。然而柑桔产业正面临着各种病虫害的威胁,其中,溃疡病和黄龙病是柑桔产业中具有毁灭性危害的细菌病害,往往给柑桔产业造成巨大的经济损失。因此,解决柑桔病害成为柑桔产业可持续发展的关键性因素,但目前世界柑桔生产中溃疡病的危害仍然没有克服,严重影响果实的品质和经济利益。
本实验前期研究表明来自柞蚕的抗菌肽基因能显著提高柑桔对溃疡病细菌的抗性,并获得了遗传稳定、抗性增加的转基因柑桔植株。在此基础上,本研究针对溃疡病细菌细胞间隙寄生的特点,人工合成分泌型抗菌肽基因,利用农杆菌介导将其转入柑桔中,使抗菌肽基因在柑桔中分泌表达。为进一步有效利用抗菌肽基因改良柑桔抗病性提供一条新的思路和材料。取得的阶段性结果如下：
1.分泌型cecropin B基因的人工合成
在参考大量国内外文献的基础上,选取了PR1a、aAM、GRP、PG、SPS、AAT等6个信号肽,通过生物信息学的分析,得到了四个具有潜在的分泌功能的信号肽,分别为PR1a、aAM、PG、AAT。从中选取了PR1a和A-AT两个信号肽序列,用于合成分泌型cecropin B基因。通过重组PCR成功获得了PR1a:CB和AAT:CB两个分泌型的cecropin B基因。
2.分泌型cecropin B基因的亚细胞定位表达分析
以红色荧光蛋白基因rfp为报告基因,构建用于抗菌肽基因亚细胞定位表达分析的植物表达载体,通过农杆菌介导法转化洋葱上表皮细胞,荧光显微镜观察抗菌肽基因的亚细胞定位表达情况。研究结果表明,PR1a和AAT信号肽均能高效促进抗菌肽基因的胞外分泌表达。
3.超量表达抗菌肽基因植物表达载体的构建及柑桔的遗传转化
以CaMV 35S启动子为抗菌肽基因的调控序列,构建了p35S—PR1a:CB、p35S-AAT:CB和p35S-CB植物表达载体,并通过农杆菌介导法将上述植物表达载体分别转化锦橙,经GUS染色和PCR技术筛选获得了70余个转基因株系。
4.抗菌肽基因在转基因植株中表达的qPCR分析
分别从不同转基因锦橙株系中提取总RNA,将其反转录为cDNA后,以actin为内参、转pGN的转基因株系为空白对照,进行qPCR反应,对转基因植株中CB基因的表达进行定量。结果表明,CB基因在转PR1a:CB.AAT:CB和CB基因的转基因株系中均有不同程度的表达。
Citrus is one of the most important commodity fruit in the world, and its plantation area and output rank first in all the kinds of fruit, and citrus fruit has become the fifth largest agricultural products in international trade. However, one of the most challenges in citrus industry is the threat of various diseases and insect pests, and Citrus canker disease is devastating disaster for citrus industry. Therefore, it becomes important to research strategies controlling the disaster of citrus canker disease.
The preliminary research showed that the antibacterial peptides from tussah could significantly improve citrus resistance to canker bacteria. Here, based on the intercellular parasitizing characteristics of citrus canker bacteria, two new editions of cecropin B gene for secreting antibacterial peptide to exacellular space were synthesized by PCR and transformed into citrus by Agrobacterium-mediated genetic transformation. This will provide a novel strategy and materials for further effectively modifying citrus resistance via antimicrobial peptides. The results are as follows:
1. The synthetic of secretory cecropin B genes
Based on associated literarures,6 selected signal peptides(PRla、aAM、GRP、PG、SPS、AAT) were subjected to bioinformatics analysis, and PRla, aAM, PG and AAT signal peptides showed the potential of improving intercellular excretion of Cecropin B protein and were used to design new cecropin B gene for targeting Cecropin B peptide to intercellular space. And finally PRla:CB and AAT.CB were successfully obtained by PCR method.
2. Sub-cellular localization expression analysis of PRla:CB and AAT:CB genes
Using the red fluorescent protein (RFP) gene as reporter gene, plant expression vectors for sub-cellular localization gene expression analysis were constructed and were transferred into onion skin via Arobacterium-mediated transformation. The sub-cellular localization expression of antibacterial peptides was observed with fluorescence microscope.The results showed that either of PR1a or AAT signal peptide could efficiently promote antimicrobial peptide molecule secreting to extracellular space of onion skin.
3. Construction of plant expression vectors with overexpressing antibacterial peptides gene and genetic transformation of citrus
CaMV 35S promoter was used to drive the expression of antibacterial peptides gene, and p35S-PRla:CB、p35S-AAT:CB and p35S-CB plant expression vectors were constructed and transferred to Citrus sinensis (Linn.) Osbeck cv. Jin Cheng by 4grobacterium-mediated method.72 transgenic plants containing antibacterial peptides gene were obtained by GUS histochemical straining and PCR analysis.
4. Real-time PCR analysis of antimicrobial peptide gene expression in transgenic plants
Total RNA were extracted from different independent transgenic Jincheng plants, and the expression levels of cecropin B in transgene plant were investigated by Real-time PCR using actin gene as reference.The result displayed that the expression of cecropin B was detected in all transgenic plants transformed by PRla:CB, AAT.CB or CB gene, while no cecropin B mRNA was found in pGN transgenic plants without cecropin B.
本实验前期研究表明来自柞蚕的抗菌肽基因能显著提高柑桔对溃疡病细菌的抗性,并获得了遗传稳定、抗性增加的转基因柑桔植株。在此基础上,本研究针对溃疡病细菌细胞间隙寄生的特点,人工合成分泌型抗菌肽基因,利用农杆菌介导将其转入柑桔中,使抗菌肽基因在柑桔中分泌表达。为进一步有效利用抗菌肽基因改良柑桔抗病性提供一条新的思路和材料。取得的阶段性结果如下：
1.分泌型cecropin B基因的人工合成
在参考大量国内外文献的基础上,选取了PR1a、aAM、GRP、PG、SPS、AAT等6个信号肽,通过生物信息学的分析,得到了四个具有潜在的分泌功能的信号肽,分别为PR1a、aAM、PG、AAT。从中选取了PR1a和A-AT两个信号肽序列,用于合成分泌型cecropin B基因。通过重组PCR成功获得了PR1a:CB和AAT:CB两个分泌型的cecropin B基因。
2.分泌型cecropin B基因的亚细胞定位表达分析
以红色荧光蛋白基因rfp为报告基因,构建用于抗菌肽基因亚细胞定位表达分析的植物表达载体,通过农杆菌介导法转化洋葱上表皮细胞,荧光显微镜观察抗菌肽基因的亚细胞定位表达情况。研究结果表明,PR1a和AAT信号肽均能高效促进抗菌肽基因的胞外分泌表达。
3.超量表达抗菌肽基因植物表达载体的构建及柑桔的遗传转化
以CaMV 35S启动子为抗菌肽基因的调控序列,构建了p35S—PR1a:CB、p35S-AAT:CB和p35S-CB植物表达载体,并通过农杆菌介导法将上述植物表达载体分别转化锦橙,经GUS染色和PCR技术筛选获得了70余个转基因株系。
4.抗菌肽基因在转基因植株中表达的qPCR分析
分别从不同转基因锦橙株系中提取总RNA,将其反转录为cDNA后,以actin为内参、转pGN的转基因株系为空白对照,进行qPCR反应,对转基因植株中CB基因的表达进行定量。结果表明,CB基因在转PR1a:CB.AAT:CB和CB基因的转基因株系中均有不同程度的表达。
Citrus is one of the most important commodity fruit in the world, and its plantation area and output rank first in all the kinds of fruit, and citrus fruit has become the fifth largest agricultural products in international trade. However, one of the most challenges in citrus industry is the threat of various diseases and insect pests, and Citrus canker disease is devastating disaster for citrus industry. Therefore, it becomes important to research strategies controlling the disaster of citrus canker disease.
The preliminary research showed that the antibacterial peptides from tussah could significantly improve citrus resistance to canker bacteria. Here, based on the intercellular parasitizing characteristics of citrus canker bacteria, two new editions of cecropin B gene for secreting antibacterial peptide to exacellular space were synthesized by PCR and transformed into citrus by Agrobacterium-mediated genetic transformation. This will provide a novel strategy and materials for further effectively modifying citrus resistance via antimicrobial peptides. The results are as follows:
1. The synthetic of secretory cecropin B genes
Based on associated literarures,6 selected signal peptides(PRla、aAM、GRP、PG、SPS、AAT) were subjected to bioinformatics analysis, and PRla, aAM, PG and AAT signal peptides showed the potential of improving intercellular excretion of Cecropin B protein and were used to design new cecropin B gene for targeting Cecropin B peptide to intercellular space. And finally PRla:CB and AAT.CB were successfully obtained by PCR method.
2. Sub-cellular localization expression analysis of PRla:CB and AAT:CB genes
Using the red fluorescent protein (RFP) gene as reporter gene, plant expression vectors for sub-cellular localization gene expression analysis were constructed and were transferred into onion skin via Arobacterium-mediated transformation. The sub-cellular localization expression of antibacterial peptides was observed with fluorescence microscope.The results showed that either of PR1a or AAT signal peptide could efficiently promote antimicrobial peptide molecule secreting to extracellular space of onion skin.
3. Construction of plant expression vectors with overexpressing antibacterial peptides gene and genetic transformation of citrus
CaMV 35S promoter was used to drive the expression of antibacterial peptides gene, and p35S-PRla:CB、p35S-AAT:CB and p35S-CB plant expression vectors were constructed and transferred to Citrus sinensis (Linn.) Osbeck cv. Jin Cheng by 4grobacterium-mediated method.72 transgenic plants containing antibacterial peptides gene were obtained by GUS histochemical straining and PCR analysis.
4. Real-time PCR analysis of antimicrobial peptide gene expression in transgenic plants
Total RNA were extracted from different independent transgenic Jincheng plants, and the expression levels of cecropin B in transgene plant were investigated by Real-time PCR using actin gene as reference.The result displayed that the expression of cecropin B was detected in all transgenic plants transformed by PRla:CB, AAT.CB or CB gene, while no cecropin B mRNA was found in pGN transgenic plants without cecropin B.
引文
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