辣椒雄性不育花器官败育特征的研究及辣椒CaCP26的cDNA克隆和分析
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摘要
以西北农林科技大学园艺学院辣椒遗传育种课题组提供的辣椒雄性不育材料HW200A～HW204A及其相应的保持系HW200B～HW204B为试材,对五组辣椒不育系和保持系的花器官性状进行了观察比较;选出三组辣椒不育系和保持系HW200A、HW201A、HW204A和HW200B、HW201B、HW204B,对这3组辣椒的小孢子发育过程进行细胞学观察;根据光学显微镜观察结果对新型败育现象HW204A及其保持系HW204B进行了透射电镜的观察,测定了不育系和保持系的花粉生活力、单个花药的花粉量,用扫描电镜观察HW204A和HW204B的成熟花粉粒的外部形态。除此之后,西北农林科技大学园艺学院辣椒遗传育种课题组利用cDNA-AFLP方法在辣椒雄性不育两用系的可育株花蕾与不育株花蕾获得阳性差异TDF(transcription derived fragment),对该基因全长进行克隆,并对其进行生物信息学分析和表达分析。主要结果如下:
1.根据花药长和宽、花朵开张度、花器官花瓣数、花柱长、花丝长、花托长和宽等6个性状进行了观察和对比,发现五组辣椒雄性不育系的花药均比相应保持系小,花丝比相应保持系短。
2.对辣椒不育系HW200A、HW201A、HW201A和其相应保持系HW200B、HW201B、HW204B的小孢子发育过程进行石蜡切片观察,发现两种不同的败育类型。特征如下:(1)HW200A,HW201A可以形成正常的四分体,绒毡层细胞极度膨胀,解体后与四分体粘在一起,使四分体无法正常发育最终导致败育;(2)HW204A可以形成正常的单核小孢子,但是此时绒毡层完全解体后分散在花粉囊腔,与小孢子连成一片,最终小孢子无法形成成熟的花粉粒。
3.雄性不育系HW204A及其保持系HW204B的电镜透射观察结果表明,雄性不育材料HW204A的小孢子败育发生在小孢子的单核靠边期,绒毡层解体后与单核小孢子粘在一起导致单核小孢子细胞核的解体,从而发生败育。
4.雄性不育系HW204A及其保持系HW204B的花粉量、花粉生活力测定结果表明,不育系的小孢子花粉量为0,花粉生活力为0;保持系的小孢子花粉量为22625±2017.11,花粉生活力为92.41%±2.63%。
5.雄性不育系HW204A及其保持系HW204B的花粉粒电子扫描结果表明,不育系的小孢子败育彻底,呈星星片状;保持系的小孢子发育成为正常的成熟的花粉粒。
6.通过电子克隆的方法,获得大小为865bp的cDNA序列,并在辣椒雄性不育两用系HW58可育株花蕾中进行了RT-PCR验证,测序结果与电子克隆结果的一致性高达99.61%。经Blast N比对,所克隆基因与烟草CP26基因序列的同源性高达90%,命名为CaCP26(GenBank登录号:GQ999612)。该基因大小为871bp,包含864bp的完整开放读码框,编码287个氨基酸,所编码的蛋白质为疏水蛋白,分子量30710.47 Da,理论等电点为5.807,有跨膜域,无信号肽,二级结构以无规则卷曲为主;利用Swiss-Model得到CaCP26蛋白(65aa-285aa)的三级结构;系统进化树结果显示,辣椒CaCP26与烟草CP26的亲缘关系最近。
7.半定量RT-PCR分析表明,CaCP26在小孢子单核期的辣椒雄性不育两用系可育株的表达量是不育株花蕾的表达量6.58倍。
The male sterile materials of pepper(HW200A, HW201A, HW202A, HW203A, HW204A) and their corresponding maintainer lines(HW200B, HW201B, HW202B, HW203B, HW204B) were provided by College of Horticulture of Northwest A & F University. Firstly I mainly compared six combinations of floral parts and made analysis. Secondly, I chose three combinations and observed the the microstructure of microspore development. According to the result of observation with light microscope, the combination named HW204A and HW204B was further observed under TEM(transmission electron microscope). I determined the pollen vitality of this combination and the number of pollen in an anther. Then morphological studies of pollen of pepper under SEM(scanning electron microscope) are observed. In addition, a positive different TDF(transcription derived fragment) was obtained from buds of the fertile individuals and the sterile individuals of genetic male sterile pepper named HW58 with cDNA-AFLP technique.The main results were obtained as follows:
1. Observing and comparing the six different floral organs, the anthers of male sterile lines are smaller than that of maintainer lines, however, the filaments of the male sterile lines are longer than that of maintainer lines.
2. Observing and analyzing the microspore developments of three combinations of male sterile lines and their corresponding maintainer lines named HW200A,HW201A,HW204A and HW200B,HW201B,HW204B with light microscope, two different types of the abortion occurred:(1)In HW200A and HW201A lines, pollen tetrads were all formed, but as the tapetal cells greatly inflated, broke down, and then sticked to the pollen tetrads. It finally could not form mononuclear microspore;(2)In HW204A line, the mononuclear microspore successfully formed, but could not form mature pollen grain.
3. After the stage of mononuclear microspore, the tapetal cells broke down, sticked to the mononuclear microspore, then the mononuclear microspore broke down itself and finally resulted in abortion in HW204 under TEM.
4. The determination of the vitality of pollen and the number of pollen in an anther: no pollen in male sterile line named HW204 with no vitality; 22625±2017.11 pollen grain in maintainer line named HW204B with 92.41%±2.63%.
5 Based on the TDF, the cDNA which is 865bp was acquired by electronic cloned method. It was verified further in buds of the fertile individuals of genetic male sterile pepper named HW58 with RT-PCR technolgy. The sequence cloned in pepper had a high identity up to 99.61% with the sequence obtained from electronic clone. The result of Blast N demonstrated that the gene shared 90% homology to the CP26 of tobacco. So we named this gene as CaCP26(GenBank accession number GQ999612). The structure, the composition and character of amino acids of CaCP26 gene sequence were analysed by bioinformatics software. The gene was 871bp in length, including a 864bp clear open reading frame which codes 287 amino acid. The results were that its relative molecular weight is 30710.47 Da, isoelectric point was 5.087, a kind of hydrophobic protein. It had three transmembrane helics, without a signal peptide. The secondary structure of CaCP26 was mainly composed of random coil. Swiss-Model software predicted a tertiary structure including 65aa-285aa. A molecular evolution tree showed that CP26 of pepper had high genetic relationship with tobacco.
6. The semi-quantitative RT-PCR was employed to analyse the expression of CaCP26 in buds of sterile individuals and fertile individuals in uninucleate stage, in which the fertile individuals exhibited higher relative expression than the sterile individuals.
1.根据花药长和宽、花朵开张度、花器官花瓣数、花柱长、花丝长、花托长和宽等6个性状进行了观察和对比,发现五组辣椒雄性不育系的花药均比相应保持系小,花丝比相应保持系短。
2.对辣椒不育系HW200A、HW201A、HW201A和其相应保持系HW200B、HW201B、HW204B的小孢子发育过程进行石蜡切片观察,发现两种不同的败育类型。特征如下:(1)HW200A,HW201A可以形成正常的四分体,绒毡层细胞极度膨胀,解体后与四分体粘在一起,使四分体无法正常发育最终导致败育;(2)HW204A可以形成正常的单核小孢子,但是此时绒毡层完全解体后分散在花粉囊腔,与小孢子连成一片,最终小孢子无法形成成熟的花粉粒。
3.雄性不育系HW204A及其保持系HW204B的电镜透射观察结果表明,雄性不育材料HW204A的小孢子败育发生在小孢子的单核靠边期,绒毡层解体后与单核小孢子粘在一起导致单核小孢子细胞核的解体,从而发生败育。
4.雄性不育系HW204A及其保持系HW204B的花粉量、花粉生活力测定结果表明,不育系的小孢子花粉量为0,花粉生活力为0;保持系的小孢子花粉量为22625±2017.11,花粉生活力为92.41%±2.63%。
5.雄性不育系HW204A及其保持系HW204B的花粉粒电子扫描结果表明,不育系的小孢子败育彻底,呈星星片状;保持系的小孢子发育成为正常的成熟的花粉粒。
6.通过电子克隆的方法,获得大小为865bp的cDNA序列,并在辣椒雄性不育两用系HW58可育株花蕾中进行了RT-PCR验证,测序结果与电子克隆结果的一致性高达99.61%。经Blast N比对,所克隆基因与烟草CP26基因序列的同源性高达90%,命名为CaCP26(GenBank登录号:GQ999612)。该基因大小为871bp,包含864bp的完整开放读码框,编码287个氨基酸,所编码的蛋白质为疏水蛋白,分子量30710.47 Da,理论等电点为5.807,有跨膜域,无信号肽,二级结构以无规则卷曲为主;利用Swiss-Model得到CaCP26蛋白(65aa-285aa)的三级结构;系统进化树结果显示,辣椒CaCP26与烟草CP26的亲缘关系最近。
7.半定量RT-PCR分析表明,CaCP26在小孢子单核期的辣椒雄性不育两用系可育株的表达量是不育株花蕾的表达量6.58倍。
The male sterile materials of pepper(HW200A, HW201A, HW202A, HW203A, HW204A) and their corresponding maintainer lines(HW200B, HW201B, HW202B, HW203B, HW204B) were provided by College of Horticulture of Northwest A & F University. Firstly I mainly compared six combinations of floral parts and made analysis. Secondly, I chose three combinations and observed the the microstructure of microspore development. According to the result of observation with light microscope, the combination named HW204A and HW204B was further observed under TEM(transmission electron microscope). I determined the pollen vitality of this combination and the number of pollen in an anther. Then morphological studies of pollen of pepper under SEM(scanning electron microscope) are observed. In addition, a positive different TDF(transcription derived fragment) was obtained from buds of the fertile individuals and the sterile individuals of genetic male sterile pepper named HW58 with cDNA-AFLP technique.The main results were obtained as follows:
1. Observing and comparing the six different floral organs, the anthers of male sterile lines are smaller than that of maintainer lines, however, the filaments of the male sterile lines are longer than that of maintainer lines.
2. Observing and analyzing the microspore developments of three combinations of male sterile lines and their corresponding maintainer lines named HW200A,HW201A,HW204A and HW200B,HW201B,HW204B with light microscope, two different types of the abortion occurred:(1)In HW200A and HW201A lines, pollen tetrads were all formed, but as the tapetal cells greatly inflated, broke down, and then sticked to the pollen tetrads. It finally could not form mononuclear microspore;(2)In HW204A line, the mononuclear microspore successfully formed, but could not form mature pollen grain.
3. After the stage of mononuclear microspore, the tapetal cells broke down, sticked to the mononuclear microspore, then the mononuclear microspore broke down itself and finally resulted in abortion in HW204 under TEM.
4. The determination of the vitality of pollen and the number of pollen in an anther: no pollen in male sterile line named HW204 with no vitality; 22625±2017.11 pollen grain in maintainer line named HW204B with 92.41%±2.63%.
5 Based on the TDF, the cDNA which is 865bp was acquired by electronic cloned method. It was verified further in buds of the fertile individuals of genetic male sterile pepper named HW58 with RT-PCR technolgy. The sequence cloned in pepper had a high identity up to 99.61% with the sequence obtained from electronic clone. The result of Blast N demonstrated that the gene shared 90% homology to the CP26 of tobacco. So we named this gene as CaCP26(GenBank accession number GQ999612). The structure, the composition and character of amino acids of CaCP26 gene sequence were analysed by bioinformatics software. The gene was 871bp in length, including a 864bp clear open reading frame which codes 287 amino acid. The results were that its relative molecular weight is 30710.47 Da, isoelectric point was 5.087, a kind of hydrophobic protein. It had three transmembrane helics, without a signal peptide. The secondary structure of CaCP26 was mainly composed of random coil. Swiss-Model software predicted a tertiary structure including 65aa-285aa. A molecular evolution tree showed that CP26 of pepper had high genetic relationship with tobacco.
6. The semi-quantitative RT-PCR was employed to analyse the expression of CaCP26 in buds of sterile individuals and fertile individuals in uninucleate stage, in which the fertile individuals exhibited higher relative expression than the sterile individuals.
引文
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