不同土壤粒径条件下长春花CesA基因的克隆与表达规律研究
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摘要
本实验以长春花幼叶为材料，探讨不同土壤粒径下，长春花叶片中与细胞壁合成相关的纤维素合成酶基因(CesA)表达规律。不同的土壤粒径对土壤空间及承载土壤溶液、水分、无机离子的影响不同。通过RT-PCR获得了473bp的CesA基因cDNA片段，Northern blotting结果表明不同的土壤粒径影响叶片中CesA基因的表达，在干旱胁迫条件相同的条件下，土壤粒径为60％A+40％B和40％A+60％B时的CesA基因表达较强，而在其它土壤粒径条件下表达差别不明显。此为，本研究分析了内源激素对纤维素合成酶基因的表达规律的影响，并进一步研究不同土壤环境下植物细胞壁的生物合成，及对培育抗土壤机械阻力的品种有重要的理论意义。
具体结果如下：
1．不同处理条件，土壤粒径和土壤紧实度，以及土壤含水率不同。
2．以不同土壤粒径调控下的长春花叶片为材料，用TRIZOL法提取RNA，通过RT-PCR法，获得了473bp的CesA基因的cDNA片段(注册号EF547376)，进行Blast比对的结果表明：与桉树的CesA4(DQ014508.1)和大麦的CesA3(AY483151.1)基因的同源性为99％。
3．运用灵敏度强，特异性高的RNA作为探针进行Northern杂交，探讨不同的土壤粒径条件下长春花叶片中细胞壁的纤维素合成酶基因的表达特征。结果表明：在相同的土壤粒径下，干旱胁迫下的CesA基因的表达要比正常水分条件下弱；在土壤粒径不同而干旱胁迫条件相同CesA基因表达不同，即在干旱胁迫条件下60％A+40％B和40％A+60％B的土壤粒径下CesA基因表达较强，与在正常的水分条件下表达差别不明显。
4．分别提取不同土壤粒径调控下的长春花叶片内源性激素ABA、IAA、GA，分析其变化规律。
5．推测不同土壤粒径调控下长春花叶片内源性激素变化规律与CesA基因表达规律的相互关系。
In this study, we used the Catharanthus Roseus as the material to discuss the cloning and characterization of cellulose synthetical gene under different grain diameter and water content of soil. We got the 476bp fragment of CesA gene by RT-PCR. The result of Northern blotting indicated that the expression of CesA gene lower under the stress of water than under the normal when the level of soil grain diameter was invariable. Under the same water stress and different soil grain diameter the expression of CesA gene was higher in the situation of 60%A＋40%B and 40%A＋60%B. when the water was normal, there was not obvious distinction. In addition, we analysised the relation between endogenesis hormone and the expression of CesA gene. This work would be a theoretical basis for the further study of Cellulose synthesis and regulation, and would be helpful to the study of plant response under stress.
The main results as fellow:
1. The grain diameter, compaction and water content of soil were distinct in different condition.
2. In this study, we purified RNA from Catharanthus Roseus with TRIZOL, and tested RNA by agarose gel. RT-PCR useing RNA as a template, we got the 476bp fragment of CesA gene. The homology of it with eucalypt, Hordeum Vulgare is 99%.
3. Northern blotting could discuss the expression of CesA gene under different grain diameter and water content of soil. We can see that, the expression of CesA gene lower under the stress of water than under the normal when the leve of soil grain diameter was invariable. Under the same water stress and different soil grain diameter the expression of CesA gene was higher in the situation of 60%A＋40%B and 40%A＋60%B. when the water was normal, there was not obvious distinction.
4. Analysised the regulations of the content of ABA、IAA、GA in different soil grain diameter.
5. Deduced the relation between endogenesis hormone and expression of CesA gene.
具体结果如下：
1．不同处理条件，土壤粒径和土壤紧实度，以及土壤含水率不同。
2．以不同土壤粒径调控下的长春花叶片为材料，用TRIZOL法提取RNA，通过RT-PCR法，获得了473bp的CesA基因的cDNA片段(注册号EF547376)，进行Blast比对的结果表明：与桉树的CesA4(DQ014508.1)和大麦的CesA3(AY483151.1)基因的同源性为99％。
3．运用灵敏度强，特异性高的RNA作为探针进行Northern杂交，探讨不同的土壤粒径条件下长春花叶片中细胞壁的纤维素合成酶基因的表达特征。结果表明：在相同的土壤粒径下，干旱胁迫下的CesA基因的表达要比正常水分条件下弱；在土壤粒径不同而干旱胁迫条件相同CesA基因表达不同，即在干旱胁迫条件下60％A+40％B和40％A+60％B的土壤粒径下CesA基因表达较强，与在正常的水分条件下表达差别不明显。
4．分别提取不同土壤粒径调控下的长春花叶片内源性激素ABA、IAA、GA，分析其变化规律。
5．推测不同土壤粒径调控下长春花叶片内源性激素变化规律与CesA基因表达规律的相互关系。
In this study, we used the Catharanthus Roseus as the material to discuss the cloning and characterization of cellulose synthetical gene under different grain diameter and water content of soil. We got the 476bp fragment of CesA gene by RT-PCR. The result of Northern blotting indicated that the expression of CesA gene lower under the stress of water than under the normal when the level of soil grain diameter was invariable. Under the same water stress and different soil grain diameter the expression of CesA gene was higher in the situation of 60%A＋40%B and 40%A＋60%B. when the water was normal, there was not obvious distinction. In addition, we analysised the relation between endogenesis hormone and the expression of CesA gene. This work would be a theoretical basis for the further study of Cellulose synthesis and regulation, and would be helpful to the study of plant response under stress.
The main results as fellow:
1. The grain diameter, compaction and water content of soil were distinct in different condition.
2. In this study, we purified RNA from Catharanthus Roseus with TRIZOL, and tested RNA by agarose gel. RT-PCR useing RNA as a template, we got the 476bp fragment of CesA gene. The homology of it with eucalypt, Hordeum Vulgare is 99%.
3. Northern blotting could discuss the expression of CesA gene under different grain diameter and water content of soil. We can see that, the expression of CesA gene lower under the stress of water than under the normal when the leve of soil grain diameter was invariable. Under the same water stress and different soil grain diameter the expression of CesA gene was higher in the situation of 60%A＋40%B and 40%A＋60%B. when the water was normal, there was not obvious distinction.
4. Analysised the regulations of the content of ABA、IAA、GA in different soil grain diameter.
5. Deduced the relation between endogenesis hormone and expression of CesA gene.
引文
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