烟草遗传多样性分析及其氮代谢的遗传操作
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摘要
烟草(Nicotiana tabacum L.)是重要的经济作物,是国民经济的重要支柱之一。目前国内用于烟草育种的亲本比较集中,种质遗传基础比较狭窄,很难培育出新型烟草品种,因此有必要研究烟草种质资源之间的遗传多样性,为今后合理选择烟草育种的亲本以及最大限度地利用烟草种质资源提供理论依据。
氮是烟草生长所需的三大营养元素之一,同时也是烟草生长的主要限制因子。氮肥施用成本占烟草生产成本的40%。在烟草氮素代谢途径中,谷氨酰胺合成酶(GS)催化铵转化成谷氨酰胺是无机氮同化成有机氮的重要步骤,同时也是氮同化作用过程的一个关键的限速步骤。植物对氮素的利用还需要大量的碳骨架即a-酮戊二酸参与,碳的水平对GS基因的表达有积极作用。而转录因子Dof1与碳代谢相关基因的表达有紧密联系。现有的研究表明利用传统育种方法选育氮素利用率增强的烟草品种很难成功,期望利用植物基因工程技术,在烟草中过量表达谷胺酰胺合成酶及与碳代谢途径相关的Dof1转录因子来提高烟草氮素利用率及其对低氮胁迫的耐受力。本论文主要获得了以下结果:
1、利用ISSR分子标记对25个烟草种质资源进行了遗传多样性分析。从50条ISSR引物中共筛选出5条引物能对烟草基因组DNA进行有效扩增。这5条引物在25份材料中共扩增出100个位点,其中有76个多态性位点,多态性比率为76%。用NTSYSpc-2.10e软件计算烟草种质间的Jaccard遗传相似系数,25种烟草种质之间的遗传相似性系数界于0.313-0.938之间,平均遗传相似性系数为0.737。野生烟与栽培烟草相似系数较低,说明它们之间存在较高的遗传多样性;利用UPGMA进行系统聚类分析,25个烟草种质资源划分成2组,3个野生烟聚成一类,22个栽培烟草种质聚成另一个大类。
2、通过PCR方法,从拟南芥中分离出细胞质型谷氨酰胺合成酶GS1基因和叶绿体型谷氨酰胺合成酶GS2基因,并成功构建了在组成型启动子CaMV35S以及光诱导型启动子1,5—二磷酸核酮糖羧化酶加氧酶小亚基RbcS控制下的组成/诱导型植物表达载体pH2-35S-PRbcS-GS1和pK2-35S-PRbcS-~*T-GS2。
3、以野生型烟草WT以及转pPZP221-PRbcS-Dof1表达载体的烟草WT-Dof1为出发材料,通过叶盘转化法,分别转入GS1和GS2组成/诱导型植物表达载体pH2-35S-PRbcS-GS1和pK2-35S-PRbcS-~*T-GS2,获得了6种转基因烟草:WT-GS1,WT-GS2,WT-Dof1.GS1,WT-Dof1-GS2,WT-GS1-GS2,WT-Dof1-GS1-GS2。进行相应的抗生素筛选,基因组PCR检测以及RNA转录水平检测,实验结果表明GS1,GS2基因已转入相应的烟草中。
4、测定7种转基因烟草的谷氨酰胺合成酶酶活,转基因烟草的酶活约为野生型对照烟草酶活的1.44—2.75倍。测定在低氮胁迫条件下生长的5种转基因烟草WT-GS2,WT-Dof1,WT-Dof1-GS1,WT-GS1-GS2,WT-Dof1-GS1-GS2与野生型烟草的苗期株高,5种转基因烟草与野生型烟草的株高比分别为:1.14,1.23,1.71,1.43,2.27,说明过量表达GS1、GS2基因和Dof1转录因子能够提高转基因烟草的GS酶活,促进其在低氮条件下的生长。
Tobacco (Nicotiana tabacum L.) is an important economic crop and also one of the important pillars of the national economy. At present, the parents for tobacco breeding are relatively concentrated and germplasm genetic base is relatively narrow. It is difficult to produce new varieties of tobacco. Therefore, it is necessary to examine germplasm resources' genetic diversity among the tobaccos for providing a theoretical basis for parents choice of the tobacco breeding and the maximum utilization of tobacco germplasm resources.
Nitrogen is one of three major nutrition elements and also one of the rate-limiting factors in tabacoo growth. Cost of nitrogen fertilization accounts for 40% of the total cost in tobacco production. Transforming ammonium into the glutamine catalyzed by Glutamine synthetase (GS) is an important step in tabacoo nitrogen metabolisim pathway. It is also a key limited-rate procedure in nitrogen assimilation. The nitrogen utilization for plant needs the participation of a large amounts of carbon skeleton (a-ketoglutarate). The level of carbon plays a positive role in GS expression. The transcription factor Dof1 is closely relation to expression of relative carbon metabolism gene. Present research shows that traditional breeding method is difficult to breed crop varieties with enhancing nitrogen utilization. So it is expected to over-express GS and Dof1 which related to carbon metabolic pathway in tobacco by using plant genetic engineering technology to improve nitrogen utilization and the tolerance to low-nitrogen stress of tobacco. This paper obtained the following results:
1. The genetic diversity was assessed among 25 tobacco accessions by ISSR marker, and five effective primers were screened out of the 50 ISSR primers. Those five primers finally amplified 100 allels, of which 76 allels were polymorphic, and the ratio of polymorphic allels was 76%. The Jaccard's genetic similarity (GS) coefficients were calculated by NTSYSpc-2.10e software. The result indicated that the GS coefficients among the 25 tobacco accessions were ranged from 0.313 to 0.938 with an average of 0.737. The GS coefficients between three wild tobacco accessions and the cultivated tobacco accessions were smaller than those among the cultivated tobacco accessions, and this suggested that the genetic variation was high between the wild and the cultivated tobacco accessions. The 25 tobacco accessions were divided into two groups on the UPGMA dendrogram constructed from GS coefficients. The three wild tobacco accessions were clustered into one group, while the 22 cultivated tobacco accessions fell into the other group.
2. GS1 and GS2 were separated from Arabidopisis cDNA by Polymerase Chain Reaction (PCR). The constitutive/inducible plant expression vectors pH2-35S-PRbcS-GS1 and pK2-35S-PRbcS-*T-GS2 which controled by CaMV35S and RbcS had been constructed.
3. These constitutive/inducible plant expression vectors pH2-35S-PRbcS-GS1 and pK2-35S-PRbcS-*T-GS2 were respectively transformed into wild type tobacco (WT) and the transgenic tobacco which had been transformed plant expression vector pPZP221-PRbcS-Dof by Agrobacterium-mediated method, we got six kinds of transgenic tobaccos: WT-GS1 , WT-GS2 , WT-Dof1-GS1 , WT-Dof1-GS2 , WT-GS1-GS2 , WT-Dof1-GS1-GS2. These transgenic tobaccos were confirmed through antibiotics selection and test of genomic PCR and RT-PCR.
4. The datas of GS activities in 7 kinds transgenic tobaccos showed that the transgenic tobaccos' GS activities were 1.44-2.75 folds compared with control plants (WT). Determine the seedling height of transgenic tobaccos WT-GS2, WT-Dof1, WT-Dof1-GS1, WT-GS1-GS2, WT-Dof1-GS1-GS2 and wild-type tobacco cultivated under the condition of low nitrogen stress, the ratios were: 1.14, 1.23, 1.71, 1.43, 2.27, respectively. These showed that overexpression of GS1, GS2 and Dof1 can improve the GS activities and promote transgenic tobaccos' growth under the condition of low-nitrogen.
氮是烟草生长所需的三大营养元素之一,同时也是烟草生长的主要限制因子。氮肥施用成本占烟草生产成本的40%。在烟草氮素代谢途径中,谷氨酰胺合成酶(GS)催化铵转化成谷氨酰胺是无机氮同化成有机氮的重要步骤,同时也是氮同化作用过程的一个关键的限速步骤。植物对氮素的利用还需要大量的碳骨架即a-酮戊二酸参与,碳的水平对GS基因的表达有积极作用。而转录因子Dof1与碳代谢相关基因的表达有紧密联系。现有的研究表明利用传统育种方法选育氮素利用率增强的烟草品种很难成功,期望利用植物基因工程技术,在烟草中过量表达谷胺酰胺合成酶及与碳代谢途径相关的Dof1转录因子来提高烟草氮素利用率及其对低氮胁迫的耐受力。本论文主要获得了以下结果:
1、利用ISSR分子标记对25个烟草种质资源进行了遗传多样性分析。从50条ISSR引物中共筛选出5条引物能对烟草基因组DNA进行有效扩增。这5条引物在25份材料中共扩增出100个位点,其中有76个多态性位点,多态性比率为76%。用NTSYSpc-2.10e软件计算烟草种质间的Jaccard遗传相似系数,25种烟草种质之间的遗传相似性系数界于0.313-0.938之间,平均遗传相似性系数为0.737。野生烟与栽培烟草相似系数较低,说明它们之间存在较高的遗传多样性;利用UPGMA进行系统聚类分析,25个烟草种质资源划分成2组,3个野生烟聚成一类,22个栽培烟草种质聚成另一个大类。
2、通过PCR方法,从拟南芥中分离出细胞质型谷氨酰胺合成酶GS1基因和叶绿体型谷氨酰胺合成酶GS2基因,并成功构建了在组成型启动子CaMV35S以及光诱导型启动子1,5—二磷酸核酮糖羧化酶加氧酶小亚基RbcS控制下的组成/诱导型植物表达载体pH2-35S-PRbcS-GS1和pK2-35S-PRbcS-~*T-GS2。
3、以野生型烟草WT以及转pPZP221-PRbcS-Dof1表达载体的烟草WT-Dof1为出发材料,通过叶盘转化法,分别转入GS1和GS2组成/诱导型植物表达载体pH2-35S-PRbcS-GS1和pK2-35S-PRbcS-~*T-GS2,获得了6种转基因烟草:WT-GS1,WT-GS2,WT-Dof1.GS1,WT-Dof1-GS2,WT-GS1-GS2,WT-Dof1-GS1-GS2。进行相应的抗生素筛选,基因组PCR检测以及RNA转录水平检测,实验结果表明GS1,GS2基因已转入相应的烟草中。
4、测定7种转基因烟草的谷氨酰胺合成酶酶活,转基因烟草的酶活约为野生型对照烟草酶活的1.44—2.75倍。测定在低氮胁迫条件下生长的5种转基因烟草WT-GS2,WT-Dof1,WT-Dof1-GS1,WT-GS1-GS2,WT-Dof1-GS1-GS2与野生型烟草的苗期株高,5种转基因烟草与野生型烟草的株高比分别为:1.14,1.23,1.71,1.43,2.27,说明过量表达GS1、GS2基因和Dof1转录因子能够提高转基因烟草的GS酶活,促进其在低氮条件下的生长。
Tobacco (Nicotiana tabacum L.) is an important economic crop and also one of the important pillars of the national economy. At present, the parents for tobacco breeding are relatively concentrated and germplasm genetic base is relatively narrow. It is difficult to produce new varieties of tobacco. Therefore, it is necessary to examine germplasm resources' genetic diversity among the tobaccos for providing a theoretical basis for parents choice of the tobacco breeding and the maximum utilization of tobacco germplasm resources.
Nitrogen is one of three major nutrition elements and also one of the rate-limiting factors in tabacoo growth. Cost of nitrogen fertilization accounts for 40% of the total cost in tobacco production. Transforming ammonium into the glutamine catalyzed by Glutamine synthetase (GS) is an important step in tabacoo nitrogen metabolisim pathway. It is also a key limited-rate procedure in nitrogen assimilation. The nitrogen utilization for plant needs the participation of a large amounts of carbon skeleton (a-ketoglutarate). The level of carbon plays a positive role in GS expression. The transcription factor Dof1 is closely relation to expression of relative carbon metabolism gene. Present research shows that traditional breeding method is difficult to breed crop varieties with enhancing nitrogen utilization. So it is expected to over-express GS and Dof1 which related to carbon metabolic pathway in tobacco by using plant genetic engineering technology to improve nitrogen utilization and the tolerance to low-nitrogen stress of tobacco. This paper obtained the following results:
1. The genetic diversity was assessed among 25 tobacco accessions by ISSR marker, and five effective primers were screened out of the 50 ISSR primers. Those five primers finally amplified 100 allels, of which 76 allels were polymorphic, and the ratio of polymorphic allels was 76%. The Jaccard's genetic similarity (GS) coefficients were calculated by NTSYSpc-2.10e software. The result indicated that the GS coefficients among the 25 tobacco accessions were ranged from 0.313 to 0.938 with an average of 0.737. The GS coefficients between three wild tobacco accessions and the cultivated tobacco accessions were smaller than those among the cultivated tobacco accessions, and this suggested that the genetic variation was high between the wild and the cultivated tobacco accessions. The 25 tobacco accessions were divided into two groups on the UPGMA dendrogram constructed from GS coefficients. The three wild tobacco accessions were clustered into one group, while the 22 cultivated tobacco accessions fell into the other group.
2. GS1 and GS2 were separated from Arabidopisis cDNA by Polymerase Chain Reaction (PCR). The constitutive/inducible plant expression vectors pH2-35S-PRbcS-GS1 and pK2-35S-PRbcS-*T-GS2 which controled by CaMV35S and RbcS had been constructed.
3. These constitutive/inducible plant expression vectors pH2-35S-PRbcS-GS1 and pK2-35S-PRbcS-*T-GS2 were respectively transformed into wild type tobacco (WT) and the transgenic tobacco which had been transformed plant expression vector pPZP221-PRbcS-Dof by Agrobacterium-mediated method, we got six kinds of transgenic tobaccos: WT-GS1 , WT-GS2 , WT-Dof1-GS1 , WT-Dof1-GS2 , WT-GS1-GS2 , WT-Dof1-GS1-GS2. These transgenic tobaccos were confirmed through antibiotics selection and test of genomic PCR and RT-PCR.
4. The datas of GS activities in 7 kinds transgenic tobaccos showed that the transgenic tobaccos' GS activities were 1.44-2.75 folds compared with control plants (WT). Determine the seedling height of transgenic tobaccos WT-GS2, WT-Dof1, WT-Dof1-GS1, WT-GS1-GS2, WT-Dof1-GS1-GS2 and wild-type tobacco cultivated under the condition of low nitrogen stress, the ratios were: 1.14, 1.23, 1.71, 1.43, 2.27, respectively. These showed that overexpression of GS1, GS2 and Dof1 can improve the GS activities and promote transgenic tobaccos' growth under the condition of low-nitrogen.
引文
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