油茶种子成熟调控蛋白基因的分离克隆及功能研究
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摘要
油茶(Camellia oleifera),又名茶籽树、油茶树,属山茶科山茶属,多年生常绿小乔木或大灌木,是原产中国的乡土树种,与油橄榄、油棕和椰子并称为世界四大木本油料树种,在世界上也惟有中国有大面积的栽培。它是我国南方重要的木本油料树种,其种子富含油酸、亚油酸和亚麻酸等不饱和脂肪酸,营养价值较高,是一种优质、保健食用植物油。油茶为花果同期,从开花到种子成熟,大约需要一年的时间,油茶种子成熟的好坏直接影响着油茶的品质和产量。油茶树生命周期长,仅仅采用传统育种方法和技术很难满足当前油茶生产化需求的优良品种的要求,因此,结合基因工程育种方法和技术来培育油茶早果、早熟优良品种,是油茶产业化发展的一个必要途径。本研究通过交错延伸PCR扩增技术,获得了油茶种子成熟调控蛋白基因(Camellia oleifera ripening regulated protein gene, CoRrp gene)的全长cDNA序列和基因组的全长DNA序列；利用半定量RT-PCR方法研究了油茶种子成熟蛋白基因在油茶各组织和不同发育期种子中的表达模式；构建了超量表达载体和siRNA表达载体并转化到野生型拟南芥植株中,研究了CoRrp基因的功能。主要研究结果如下：
1.油茶种子成熟调控蛋白基因(CoRrp gene) cDNA序列的克隆
以油茶cDNA文库和EST文库为基础,分离克隆了一条油茶种子成熟调控蛋白基因的全长cDNA序列。该cDNA全长为966bp,开放阅读框含有702 bp的核苷,编码233个氨基酸的亲水多肽。生物信息学分析表明：该基因的分子量为25267.46 Da,等电点(PI)为4.43,翻译的氨基酸序列具有与番茄和马铃薯成熟调控蛋白相同的D-片段DDDDDDDVD序列和E-片段EETEAEE序列特征,该肽链中极性氨基酸比率占53.16%,其中天冬氨酸、谷氨酸、丝氨酸、苏氨酸等酸性氨基酸含量比较高,是一个半膜结合结构域,非分泌性蛋白。蛋白质高级结构中以α螺旋为主要结构元件,不规则卷曲则散布于整个蛋白质中。
2.油茶种子成熟调控蛋白基因的基因组DNA全长扩增
通过交错延伸PCR扩增技术获得了油茶种子成熟调控蛋白基因的基因组的全长DNA序列,该基因含有1个内含子,其长度为139 bp,分析该基因内含子序列发现：A+T碱基组成为69.78%,G+C碱基组成为30.22%,属于GT-AG型内含子,序列中存在一个类似于启动子(TATAA))框结构。该基因的内含子中具有真核mRNA内含子结构特征序列的5'剪接位点具有AG/GTAAGT保守序列和3'剪接位点具有TGCAG/G的保守序列。真核mRNA内含子已成为提高转基因生物外源基因表达的重要元件之一。因此,油茶种子成熟调控蛋白基因内含子的扩增为该基因的下一步超量表达研究奠定了基础。
3.油茶种子成熟调控蛋白基因在油茶各组织和不同发育期种子中的表达模式
利用半定量RT-PCR方法研究了油茶种子成熟蛋白基因在油茶根、茎、叶、花、芽和不同发育期油茶种子中的表达模式,结果显示油茶CoRrp基因的表达具有空间上的特异性,主要是在种子中表达,而在根、茎、叶中不表达。从种子的发育时期来看,CoRrp基因的表达又具有时间上的特异性,它在最早分化出来的胚中表达量较少,随着种子的发育成熟,CoRrp基因的表达水平逐渐升高,种子成熟时,CoRrp基因的表达量达到最高值。由此看出,油茶种子成熟调控蛋白基因在种子成熟过程中起着重要的作用。
4.超量表达载体和小干扰RNA表达载体的构建及转基因植株的获得
以拟南芥AT2G18110基因的CDS序列及油茶CoRrp基因的保守序列为基础,按照Tuschl设计原则,设计了小干扰RNA序列,并把它构建到中间载体pCAMBIA1304-35S中,获得了小干扰RNA表达载体pCAMBIA1304-siRNA;本研究还利用CoRrp基因基因组DNA全长序列,将CoRrp的含有内含子的完整DNA序列克隆到中间载体pCAMBIA1304-35S的CaMV 35S启动子下方的MCS中,构建了超量表达载体pCAMBIA1304-CoRrp。将构建好的超量表达载体pCAMBIA1304-CoRrp和小干扰RNA表达载体pCAMBIA1304-siRNA,用农杆菌介导Floral Dip法转化到野生型拟南芥中,获得了43株转pCAMBIA1304-CoRrp的T2代拟南芥和31株转pCAMBIA1304-siRNA的T2代拟南芥。
5.超量表达转基因拟南芥和RNA干扰表达转基因拟南芥中目的基因的表达研究
利用半定量RT-PCR检测转基因拟南芥和野生型拟南芥中(CoRrp基因的表达量,结果表明,转基因拟南芥中CoRrp的表达水平与野生型有明显差异,所挑选的12株超量表达转基因植株中CoRrp的表达水平都比野生型拟南芥中的要高；所挑选的12株干扰表达转基因植株中CoRrp的表达水平比野生型拟南芥中的表达要低,与野生型拟南芥中CoRrp的表达量和内参基因Actin的表达量相比较,10株转pCAMBIA1304-siRNA拟南芥的CoRrp的表达量只有对照的50%。
6. CoRrp在过量表达转基因拟南芥与RNA干扰表达转基因拟南芥中种子成熟调控
对超量表达转基因拟南芥与RNA干扰表达转基因拟南芥的表型观察研究发现,超量表达转基因拟南芥和RNA干扰表达转基因拟南芥与野生型拟南芥在营养生长阶段没有很大的区别,但进入生殖生长阶段时,多数超量表达转基因拟南芥的种子提前成熟,成熟度较高种子比较饱满；多数RNA干扰表达转基因拟南芥的种子推迟成熟,成熟度较低。依据统计分析结果,过量表达的T2代拟南芥中55.8%表现为种子提前成熟3-4d；RNA干扰表达的T2代拟南芥中64.5%表现为种子推迟成熟4-5d。这表明油茶CoRrp基因在种子成熟过程中具有成熟调控功能。
油茶种子成熟调控蛋白基因的表达模式及其功能研究,将为油茶的分子育种和培育早果早熟油茶新品种奠定物质和技术基础,具有重要的参考意义和应用价值。种子成熟期的提前,缩短了生长周期,也为其它科研成果在油茶上能够得到迅速应用奠定了基础。
Tea-oil tree (Camellia oleifera), Tea-oil, belongs to Camellia, and is perennial small evergreen tree or large shrub, the original Chinese native tree. Tea-oil (Camellia oleifera), olive (Olea europaea), oil palm (Elaeis quineensis), and Coconut (Cocos nucifera) are known as the four woody oleiferous plants. Tea-oil tree is one of the most important ligneous edible oil trees in China. It is the only large cultivated area in the world.90% of its seed-oil is unsaturated fatty acid, such as oleic acid, linolaic acid, linolenic acid and so on. So it is a kind of excellent vegetable oil. It needs about a year from flower to mature in its life as well as the same stage of flower and fruit, so it has the long-life development period. The trade methods and techniques are hardly satisfied requirement of excellent varieties in tea-oil industrialization project. Therefore, together with gene engineering approaches and technologies, it is necessary for breeding the excellent varieties of early-fruit, and early matures. This paper make use of the Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and the full-legnth DNA sequence of Camellia oleifera ripening regulated protein gene (CoRrp) and its expression patterns were studied, and the overexpression and small RNAi vectors were also constructed and transformed into the wild Arabidopsis thaliana, the major research conclusions are listed as follows.
1. Isolation and cloning of the CoRrp gene Based on the constructed cDNA library and EST library by the Key Lab of Non-wood Forest Product of State Forestry Administration, Central South University of Forestry and Technology, and the C. oleifera ripening-regulated protein gene was firstly and successfully isolated and cloned. And its size of full-length cDNA is 1105 bp containing an open reading frame (ORF) of 966 bp with 5'and 3'untranslated regions (UTRs) and a long Poly (A) tail, designated as CoRrp (GenBank access no. FJ713027). It encodes a 25.27-kDa protein of 233 amino acid residues with the isoelectric point (PI) of 4.43. It shares the same sequence characteristics of the D-fragment DDDDDDDVD of E-fragment with that of tomato and potato, and amini acid comes to 53.16%, in which the content of Acid amino acids, i.g., Asparagine, Glutamine, Serine and Threonine, are high, and it is a half-membrane structure and non-secretory protein. Alpha helices are the main elements of the protein, and irregular coils disperse in the whole protein.
2. The cloning of intron for the CoRrp gene The intron was obtained by PCR, and its length was 139 bp. The analysis result showed that the content of A+T is 69.78%, and of G+C is 30.22% in the intron, and belong to the intron of GT-AG, in which'there exists a similar promoter structure. The obtaining of the intron for the CoRrp gene can lay a basis for the further study of the gene overexpression.
3. The study of the expression pattern of the CoRrp gene The expression differences among roots, stems, leaves, buds, and seeds of the different developing stages were analyzed by the semi-quantitative RT-PCR, and the results showed that there were plenty of expression in seeds, trace in buds, and none in roots, stems, leaves. The expression of the CoRrp gene varied with the stages of the seed development. The expression of the CoRrp gene has the spatial characteristics, and mainly expresses in seeds, and none expression in roots, stems, and leaves. On the other hand, the expression of the CoRrp gene also has temporal characteristics from the developing stages of seeds. There is tiny amount in the initial differentiate embryo, and when the seeds gradually bulging, the expression of CoRrp gene was gradually increasing from the sixth to seventh month of a year; in the eighth to nineth month, the ripping stage of C. oleifera, the expression of the CoRrp gene dramatic increased, and reached the top in the 10th month. And the results show that the CoRrp protein play important roles in the course of the seed mature.
4. The constructed of overexpression vector and small RNA interference vector, and acquisition of transformed seedlings On the basis of the CDS sequence of AT2G18110 gene of Arabidopsis thaliana and the conserves sequence of the CoRrp gene, according to the principle of Tuschl, small RNA interference (sRNAi) was desgined, and constrcuted into the voctor pCAMBIA1304-35S, and the sRNAi expression vector was obtained; this study the genomic DNA sequence with intron CoRrp also was employed, and cloned into MCS of the down stream of CaMV 35S promoter of the vector pCAMBIA1304-35S. and the overexpression vector pCAMBIA1304-CoRrp was conducted by agrobacterium with the floral dip method, and transformed into Arabidopsis thaliana. And 43 of transformed pCAMBIA1304-CoRrp and 31 of transformed pCAMBIA1304-siRNA Arabidopsis thaliana seedlings were obtained.
5. Overexpression of transformed Arabidopis thaliana and interference expression of the target gene in transformed Arabidopsis thaliana
The expression of target gene in the transformed Arabidopsis thaliana seedlings was detected with the semi-quantitative RT-PCR, and the results showed that there were significant differences between the transformed and the wild Arabidopsis thaliana seedlings. The expression of the target gene in the 12-selected-overexpression transformed-seedlings was much higher than that in the wild Arabidopsis thaliana seedlings. On the other hand, the expression of the target gene in the 12-selected-RNAi transformed-seedlings was much lower than that in the wild Arabidopsis thaliana seedlings. Compare with the expression of CoRrp and internal control gene Actin in the wild Arabidopsis thaliana seedlings, the expression of the 10-transformed pCAMBIA1304-siRNA is only half of the control seedlings.
6. The regulation the CoRrp gene of in the seed mature of overexpression and RNA interference of transformed Arabidopsis thaliana
There was no significant difference in the overexpression and RNA interference of transformed Arabidopsis thaliana in the stage of nutrition growth, however, most of overexpression seeds ripe ahead of the normal, and of RNA interference of transformed Arabidopsis thaliana delay mature. According to the statistical results,43 seedlings (T2) of pCAMBIA1304-CoRrp Arabidopsis thaliana were obtained, of which 55.8% were matured three to four days ahead of the normal. And 31 seedlings of the pCAMBIA1304-siRNA transformed Arabidopsis thaliana were obtained, which of 64.5% were matured four to five days behind of the normal. Overall these results present that the ripening-regulated protein gene might play significant regulated roles in the C. oleifera seeds.
Overall, the research of the expression pattern and function for the CoRrp gene will lay material and technique basis for the molecular breeding and cultivating new early fruit and early mature varieties, and thus possess significant meaning and application value.
1.油茶种子成熟调控蛋白基因(CoRrp gene) cDNA序列的克隆
以油茶cDNA文库和EST文库为基础,分离克隆了一条油茶种子成熟调控蛋白基因的全长cDNA序列。该cDNA全长为966bp,开放阅读框含有702 bp的核苷,编码233个氨基酸的亲水多肽。生物信息学分析表明：该基因的分子量为25267.46 Da,等电点(PI)为4.43,翻译的氨基酸序列具有与番茄和马铃薯成熟调控蛋白相同的D-片段DDDDDDDVD序列和E-片段EETEAEE序列特征,该肽链中极性氨基酸比率占53.16%,其中天冬氨酸、谷氨酸、丝氨酸、苏氨酸等酸性氨基酸含量比较高,是一个半膜结合结构域,非分泌性蛋白。蛋白质高级结构中以α螺旋为主要结构元件,不规则卷曲则散布于整个蛋白质中。
2.油茶种子成熟调控蛋白基因的基因组DNA全长扩增
通过交错延伸PCR扩增技术获得了油茶种子成熟调控蛋白基因的基因组的全长DNA序列,该基因含有1个内含子,其长度为139 bp,分析该基因内含子序列发现：A+T碱基组成为69.78%,G+C碱基组成为30.22%,属于GT-AG型内含子,序列中存在一个类似于启动子(TATAA))框结构。该基因的内含子中具有真核mRNA内含子结构特征序列的5'剪接位点具有AG/GTAAGT保守序列和3'剪接位点具有TGCAG/G的保守序列。真核mRNA内含子已成为提高转基因生物外源基因表达的重要元件之一。因此,油茶种子成熟调控蛋白基因内含子的扩增为该基因的下一步超量表达研究奠定了基础。
3.油茶种子成熟调控蛋白基因在油茶各组织和不同发育期种子中的表达模式
利用半定量RT-PCR方法研究了油茶种子成熟蛋白基因在油茶根、茎、叶、花、芽和不同发育期油茶种子中的表达模式,结果显示油茶CoRrp基因的表达具有空间上的特异性,主要是在种子中表达,而在根、茎、叶中不表达。从种子的发育时期来看,CoRrp基因的表达又具有时间上的特异性,它在最早分化出来的胚中表达量较少,随着种子的发育成熟,CoRrp基因的表达水平逐渐升高,种子成熟时,CoRrp基因的表达量达到最高值。由此看出,油茶种子成熟调控蛋白基因在种子成熟过程中起着重要的作用。
4.超量表达载体和小干扰RNA表达载体的构建及转基因植株的获得
以拟南芥AT2G18110基因的CDS序列及油茶CoRrp基因的保守序列为基础,按照Tuschl设计原则,设计了小干扰RNA序列,并把它构建到中间载体pCAMBIA1304-35S中,获得了小干扰RNA表达载体pCAMBIA1304-siRNA;本研究还利用CoRrp基因基因组DNA全长序列,将CoRrp的含有内含子的完整DNA序列克隆到中间载体pCAMBIA1304-35S的CaMV 35S启动子下方的MCS中,构建了超量表达载体pCAMBIA1304-CoRrp。将构建好的超量表达载体pCAMBIA1304-CoRrp和小干扰RNA表达载体pCAMBIA1304-siRNA,用农杆菌介导Floral Dip法转化到野生型拟南芥中,获得了43株转pCAMBIA1304-CoRrp的T2代拟南芥和31株转pCAMBIA1304-siRNA的T2代拟南芥。
5.超量表达转基因拟南芥和RNA干扰表达转基因拟南芥中目的基因的表达研究
利用半定量RT-PCR检测转基因拟南芥和野生型拟南芥中(CoRrp基因的表达量,结果表明,转基因拟南芥中CoRrp的表达水平与野生型有明显差异,所挑选的12株超量表达转基因植株中CoRrp的表达水平都比野生型拟南芥中的要高；所挑选的12株干扰表达转基因植株中CoRrp的表达水平比野生型拟南芥中的表达要低,与野生型拟南芥中CoRrp的表达量和内参基因Actin的表达量相比较,10株转pCAMBIA1304-siRNA拟南芥的CoRrp的表达量只有对照的50%。
6. CoRrp在过量表达转基因拟南芥与RNA干扰表达转基因拟南芥中种子成熟调控
对超量表达转基因拟南芥与RNA干扰表达转基因拟南芥的表型观察研究发现,超量表达转基因拟南芥和RNA干扰表达转基因拟南芥与野生型拟南芥在营养生长阶段没有很大的区别,但进入生殖生长阶段时,多数超量表达转基因拟南芥的种子提前成熟,成熟度较高种子比较饱满；多数RNA干扰表达转基因拟南芥的种子推迟成熟,成熟度较低。依据统计分析结果,过量表达的T2代拟南芥中55.8%表现为种子提前成熟3-4d；RNA干扰表达的T2代拟南芥中64.5%表现为种子推迟成熟4-5d。这表明油茶CoRrp基因在种子成熟过程中具有成熟调控功能。
油茶种子成熟调控蛋白基因的表达模式及其功能研究,将为油茶的分子育种和培育早果早熟油茶新品种奠定物质和技术基础,具有重要的参考意义和应用价值。种子成熟期的提前,缩短了生长周期,也为其它科研成果在油茶上能够得到迅速应用奠定了基础。
Tea-oil tree (Camellia oleifera), Tea-oil, belongs to Camellia, and is perennial small evergreen tree or large shrub, the original Chinese native tree. Tea-oil (Camellia oleifera), olive (Olea europaea), oil palm (Elaeis quineensis), and Coconut (Cocos nucifera) are known as the four woody oleiferous plants. Tea-oil tree is one of the most important ligneous edible oil trees in China. It is the only large cultivated area in the world.90% of its seed-oil is unsaturated fatty acid, such as oleic acid, linolaic acid, linolenic acid and so on. So it is a kind of excellent vegetable oil. It needs about a year from flower to mature in its life as well as the same stage of flower and fruit, so it has the long-life development period. The trade methods and techniques are hardly satisfied requirement of excellent varieties in tea-oil industrialization project. Therefore, together with gene engineering approaches and technologies, it is necessary for breeding the excellent varieties of early-fruit, and early matures. This paper make use of the Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and the full-legnth DNA sequence of Camellia oleifera ripening regulated protein gene (CoRrp) and its expression patterns were studied, and the overexpression and small RNAi vectors were also constructed and transformed into the wild Arabidopsis thaliana, the major research conclusions are listed as follows.
1. Isolation and cloning of the CoRrp gene Based on the constructed cDNA library and EST library by the Key Lab of Non-wood Forest Product of State Forestry Administration, Central South University of Forestry and Technology, and the C. oleifera ripening-regulated protein gene was firstly and successfully isolated and cloned. And its size of full-length cDNA is 1105 bp containing an open reading frame (ORF) of 966 bp with 5'and 3'untranslated regions (UTRs) and a long Poly (A) tail, designated as CoRrp (GenBank access no. FJ713027). It encodes a 25.27-kDa protein of 233 amino acid residues with the isoelectric point (PI) of 4.43. It shares the same sequence characteristics of the D-fragment DDDDDDDVD of E-fragment with that of tomato and potato, and amini acid comes to 53.16%, in which the content of Acid amino acids, i.g., Asparagine, Glutamine, Serine and Threonine, are high, and it is a half-membrane structure and non-secretory protein. Alpha helices are the main elements of the protein, and irregular coils disperse in the whole protein.
2. The cloning of intron for the CoRrp gene The intron was obtained by PCR, and its length was 139 bp. The analysis result showed that the content of A+T is 69.78%, and of G+C is 30.22% in the intron, and belong to the intron of GT-AG, in which'there exists a similar promoter structure. The obtaining of the intron for the CoRrp gene can lay a basis for the further study of the gene overexpression.
3. The study of the expression pattern of the CoRrp gene The expression differences among roots, stems, leaves, buds, and seeds of the different developing stages were analyzed by the semi-quantitative RT-PCR, and the results showed that there were plenty of expression in seeds, trace in buds, and none in roots, stems, leaves. The expression of the CoRrp gene varied with the stages of the seed development. The expression of the CoRrp gene has the spatial characteristics, and mainly expresses in seeds, and none expression in roots, stems, and leaves. On the other hand, the expression of the CoRrp gene also has temporal characteristics from the developing stages of seeds. There is tiny amount in the initial differentiate embryo, and when the seeds gradually bulging, the expression of CoRrp gene was gradually increasing from the sixth to seventh month of a year; in the eighth to nineth month, the ripping stage of C. oleifera, the expression of the CoRrp gene dramatic increased, and reached the top in the 10th month. And the results show that the CoRrp protein play important roles in the course of the seed mature.
4. The constructed of overexpression vector and small RNA interference vector, and acquisition of transformed seedlings On the basis of the CDS sequence of AT2G18110 gene of Arabidopsis thaliana and the conserves sequence of the CoRrp gene, according to the principle of Tuschl, small RNA interference (sRNAi) was desgined, and constrcuted into the voctor pCAMBIA1304-35S, and the sRNAi expression vector was obtained; this study the genomic DNA sequence with intron CoRrp also was employed, and cloned into MCS of the down stream of CaMV 35S promoter of the vector pCAMBIA1304-35S. and the overexpression vector pCAMBIA1304-CoRrp was conducted by agrobacterium with the floral dip method, and transformed into Arabidopsis thaliana. And 43 of transformed pCAMBIA1304-CoRrp and 31 of transformed pCAMBIA1304-siRNA Arabidopsis thaliana seedlings were obtained.
5. Overexpression of transformed Arabidopis thaliana and interference expression of the target gene in transformed Arabidopsis thaliana
The expression of target gene in the transformed Arabidopsis thaliana seedlings was detected with the semi-quantitative RT-PCR, and the results showed that there were significant differences between the transformed and the wild Arabidopsis thaliana seedlings. The expression of the target gene in the 12-selected-overexpression transformed-seedlings was much higher than that in the wild Arabidopsis thaliana seedlings. On the other hand, the expression of the target gene in the 12-selected-RNAi transformed-seedlings was much lower than that in the wild Arabidopsis thaliana seedlings. Compare with the expression of CoRrp and internal control gene Actin in the wild Arabidopsis thaliana seedlings, the expression of the 10-transformed pCAMBIA1304-siRNA is only half of the control seedlings.
6. The regulation the CoRrp gene of in the seed mature of overexpression and RNA interference of transformed Arabidopsis thaliana
There was no significant difference in the overexpression and RNA interference of transformed Arabidopsis thaliana in the stage of nutrition growth, however, most of overexpression seeds ripe ahead of the normal, and of RNA interference of transformed Arabidopsis thaliana delay mature. According to the statistical results,43 seedlings (T2) of pCAMBIA1304-CoRrp Arabidopsis thaliana were obtained, of which 55.8% were matured three to four days ahead of the normal. And 31 seedlings of the pCAMBIA1304-siRNA transformed Arabidopsis thaliana were obtained, which of 64.5% were matured four to five days behind of the normal. Overall these results present that the ripening-regulated protein gene might play significant regulated roles in the C. oleifera seeds.
Overall, the research of the expression pattern and function for the CoRrp gene will lay material and technique basis for the molecular breeding and cultivating new early fruit and early mature varieties, and thus possess significant meaning and application value.
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