花生（Arachis hypogaea）Ara h 1基因启动子的克隆及贮藏蛋白基因Fiber-FISH的研究
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摘要
花生是一种重要的油料作物，对于花生种子发育相关基因的研究，特别是对有关储藏物质积累过程调控机制的研究具有重要的生产实践意义。同时花生又是一种植物蛋白源，对花生贮藏蛋白基因结构以及表达调控的研究，为改善花生品质，培育优质花生品种以及生产低变应原花生提供理论基础。
本实验通过用一对特异性引物，PCR扩增得到花生(Arachis hypogaea)主要致敏蛋白Ara h 1的基因的启动子片段1957bp，其序列组成与已发表序列基本一致。用其替换pBIN 35S-mGFP4载体中的35S启动子部分，组成一种花生转基因表达载体pGA1。该载体含有种子特异表达调控元件、增强表达元件和一些与转录因子结合的调控元件，是一个强启动子。此载体便于将外源基因转入花生，实现外源基因在花生中的表达，为建立花生生物反应器奠定了基础。
本文还对花生贮藏蛋白基因在基因组DNA纤维上的排布规律进行了初步探索。实验通过DNA纤维荧光原位杂交技术(Fiber-FISH)，利用花生中两个贮藏蛋白基因(Ara h 1和Ara h 3)的启动子片断为探针，对花生基因组DNA进行双色杂交。研究发现，花生贮藏蛋白基因有可能成簇分布在染色体的某个区域，连续的11个簇最长跨越了纤维上86.78μm的长度，对应的DNA长为217.82kb，簇之间间隔的平均纤维长度为3.94±0.78μm，相当于DNA长度在7.93kb-11.85kb之间。对多个双色杂交信号进行统计，发现每个簇的平均长度为5.7±1.32μm，对应的DNA长度在10.99—17.62kb之间。
Peanut is an important oil crops. The research on genes related to development of peanut seeds, especially on regulation mechanism of accumulation process of the storage materials establishes a foundation to the application in agricultural production. Peanut is also a source of plant protein for nutrition. The study of the gene structure and expression regulation of peanut storage protein provides an academic base on improving the quality of peanut, breeding peanut variety with high quality and producing hypoallergenic peanuts.
The promoter of the gene encoding the major peanut allergy protein Ara h 1 has been cloned with a pair of special primers using Polymerase Chain Reaction technique (PCR). The promoter we have got is 1957 bp. Basically, it is consistent with the sequence published. By replacing the 35S promoter in pBIN 35S-mGFP4 vector with this promoter, we have constructed a plant transgenic expression vector of peanut named pGA1. It contains seed specifically expressed elements, enhancers and some elements regulated by transcription factors. Thus the heterologous gene can be transferred to peanut and expresses in seed easily. The research lays a foundation for establishing the peanut bioreactor.
We have also done some initiative researches on the arrangement of gene family encoding peanut storage proteins. We use the technique of fiber fluorescent in situ hybridization (Fiber-FISH) and two promoters of peanut storage protein genes-Arah1 and Arah3 as probes in order to find the DNA region in peanut genome which hybridizes to the two probes. The results indicate that the genes encoding peanut storage protein may exist as some clusters in the peanut chromosomes. The longest continuous fragment contains eleven clusters and covering a 86.78 micrometer region on the DNA fiber corresponding to 217.82 kilo base pairs on the DNA sequence. The average length of the intervals among these eleven clusters is 3.94 micrometer with variance of 0.78 micrometer corresponding to 7.93 kilo base pairs to 11.85 kilo base pairs on the DNA sequence. Based on a large amount of analysises of the dual-colored hybridization signals, we have found that the average length of each cluster was 5.7 micrometer with variance of 1.32 micrometer corresponding to 10.99 kilo base pairs to 17.62 kilo base pairs on the DNA sequence.
本实验通过用一对特异性引物，PCR扩增得到花生(Arachis hypogaea)主要致敏蛋白Ara h 1的基因的启动子片段1957bp，其序列组成与已发表序列基本一致。用其替换pBIN 35S-mGFP4载体中的35S启动子部分，组成一种花生转基因表达载体pGA1。该载体含有种子特异表达调控元件、增强表达元件和一些与转录因子结合的调控元件，是一个强启动子。此载体便于将外源基因转入花生，实现外源基因在花生中的表达，为建立花生生物反应器奠定了基础。
本文还对花生贮藏蛋白基因在基因组DNA纤维上的排布规律进行了初步探索。实验通过DNA纤维荧光原位杂交技术(Fiber-FISH)，利用花生中两个贮藏蛋白基因(Ara h 1和Ara h 3)的启动子片断为探针，对花生基因组DNA进行双色杂交。研究发现，花生贮藏蛋白基因有可能成簇分布在染色体的某个区域，连续的11个簇最长跨越了纤维上86.78μm的长度，对应的DNA长为217.82kb，簇之间间隔的平均纤维长度为3.94±0.78μm，相当于DNA长度在7.93kb-11.85kb之间。对多个双色杂交信号进行统计，发现每个簇的平均长度为5.7±1.32μm，对应的DNA长度在10.99—17.62kb之间。
Peanut is an important oil crops. The research on genes related to development of peanut seeds, especially on regulation mechanism of accumulation process of the storage materials establishes a foundation to the application in agricultural production. Peanut is also a source of plant protein for nutrition. The study of the gene structure and expression regulation of peanut storage protein provides an academic base on improving the quality of peanut, breeding peanut variety with high quality and producing hypoallergenic peanuts.
The promoter of the gene encoding the major peanut allergy protein Ara h 1 has been cloned with a pair of special primers using Polymerase Chain Reaction technique (PCR). The promoter we have got is 1957 bp. Basically, it is consistent with the sequence published. By replacing the 35S promoter in pBIN 35S-mGFP4 vector with this promoter, we have constructed a plant transgenic expression vector of peanut named pGA1. It contains seed specifically expressed elements, enhancers and some elements regulated by transcription factors. Thus the heterologous gene can be transferred to peanut and expresses in seed easily. The research lays a foundation for establishing the peanut bioreactor.
We have also done some initiative researches on the arrangement of gene family encoding peanut storage proteins. We use the technique of fiber fluorescent in situ hybridization (Fiber-FISH) and two promoters of peanut storage protein genes-Arah1 and Arah3 as probes in order to find the DNA region in peanut genome which hybridizes to the two probes. The results indicate that the genes encoding peanut storage protein may exist as some clusters in the peanut chromosomes. The longest continuous fragment contains eleven clusters and covering a 86.78 micrometer region on the DNA fiber corresponding to 217.82 kilo base pairs on the DNA sequence. The average length of the intervals among these eleven clusters is 3.94 micrometer with variance of 0.78 micrometer corresponding to 7.93 kilo base pairs to 11.85 kilo base pairs on the DNA sequence. Based on a large amount of analysises of the dual-colored hybridization signals, we have found that the average length of each cluster was 5.7 micrometer with variance of 1.32 micrometer corresponding to 10.99 kilo base pairs to 17.62 kilo base pairs on the DNA sequence.
引文
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