苹果属无融合生殖相关SERK基因克隆与表达分析及遗传转化的研究
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摘要
平邑甜茶(Malus hupehensis Rehd. var. pingyiensis Jiang)是蔷薇科(Rosaceae)苹果属(Malus)湖北海棠(Malus hupehensis (Pamp.) Rehd.)的一个变种,是典型的无融合生殖型三倍体植物。平邑甜茶具有高度的无融合生殖能力,是无融合生殖型矮化砧木育种的重要母本材料。由于苹果属无融合生殖植物多为兼性无融合生殖,利用这一特点课题组前期以平邑甜茶和扎矮山定子(M. baccata)为亲本进行了有性杂交,获得了四倍性皱叶矮生型杂种后代,经鉴定这些杂种后代的无融合生殖能力显著下降。无融合生殖类型的杂种后代为什么在无融合生殖率上显著下降及其下降原因是否因某种基因调控等分子机理方面的研究国内外鲜有报道。
本研究以苹果属三倍性平邑甜茶和四倍性杂种后代株系为试材,克隆无融合生殖相关SERK基因,分析无融合生殖基因在生殖过程中的表达差异,并通过构建植物表达载体及遗传转化的研究,以期从分子水平探讨苹果属无融合生殖的分子机理。主要研究结果如下：
1.采用同源克隆的方法分别从平邑甜茶和杂种后代33#株系基因组DNA中克隆得到4个SERK同源基因家族片段,其长度分别为705bp、671bp、504bp、740bp;705bp、500bp、639bp、675bp。它们的结构基本相同,都具有一个内含子,与其他植物的SERK同源基因具有较高的同源性。
2.利用已公布的苹果基因组序列从平邑甜茶和杂种后代33#株系中分离出了SERK1和SERK4基因的cDNA全长序列,分别将其命名为MhSERK1、MhdSERK1和MhSERK4、MhdSERK4。其中MhSERK1、MhdSERK1的cDNA长度为1899bp、1881bp,分别编码632、626个氨基酸。比对MhSERK1和MhdSERK1的cDNA序列在696-714bp处存在18bp的缺失差异,分析这段差异可能与其无融合生殖能力有关。MhSERK4、 MhdSERK4的两条片段长度均为1836bp,同源性达到99.40%,分别编码611个氨基酸。氨基酸序列与棕榈科植物椰子同源性最高,达到90%以上；氨基酸编码蛋白激酶结构域高度保守,具有SERK类家族典型的LRR-RLKs结构特点。
3.根据已获得的cDNA序列,设计引物扩增仅得到了MhSERK1和MhdSERK1的DNA编码区全长序列。MhSERK1和MhdSERK1DNA编码区序列全长分别为6886bp、6719bp,均有11个外显子,10个内含子,MhSERK1和MhdSERK1的内含子分布与龙眼(HM773391)和番木瓜(EF661025)等同源基因类似,都具有11个外显子和10个内含子结构。
4.利用实时定量RT-PCR的方法分别检测了SERK基因在三倍性平邑甜茶和四倍性杂种后代33#株系不同器官、组织及子房发育不同时期的表达情况o SERK1、SERK2、 SERK3、SERK4基因主要在生殖器官中表达,而且在生殖器官中的表达平邑甜茶明显高于杂种后代33#,说明SERK基因是与无融合生殖相关的基因；在不同花期子房的表达分析SERK1在平邑甜茶花蕾期(花前5天)子房中表达量最高,其次是盛花期,盛花期后(花后5天)的子房中表达量最低；而在杂种后代中盛花期后的表达量最高,其次是花蕾期,在盛花期表达量最低。SERK1无论在平邑甜茶还是在杂种后代33#的子房中都是表达量最高,其次是在雄蕊中；而在平邑甜茶花瓣中表达量最低,在杂种后代33#的叶片、花瓣和雌蕊中表达量都很低,几乎不表达。SERK2基因在平邑甜茶开花后第2d的子房中表达量最高,大约是杂种后代33#表达量的5倍；而在杂种后代33#开花后第3d的表达量最高,大约是平邑甜茶表达量的二倍。SERK3基因在平邑甜茶开花后第2d子房中的表达量最高,大约是杂种后代33#表达量的15倍；SERK4基因在平邑甜茶开花后第1d的表达量最高,而且在平邑甜茶花后1-5d的表达量均高于杂种后代33#株系。
5.构建了MhSERK1、MhdSERK1和MhSERK4、MhdSERK4植物表达载体,将其分别命名为pBI121-MhSERK1、pBI121-MhdSERK1和pBI121-MhSERK4、 pBI121-MhdSERK4,并成功导入农杆菌菌株EHA105。
6.通过农杆菌侵染法,利用构建好的pBI121-MhSERK1、pBI121-MhdSERKl植物表达载体,分别以Micro-Tom番茄和烟草为试材进行遗传转化研究,共获得7株和6株烟草抗性植株,通过PCR检测验证转化成功。
Pingyi Tiancha(Malus hupehensis Rehd. var. pingyiensis Jiang), a variety of crabapple (Malus hupehensis (Pamp.) Rehd.), which belongs to the Malus of Rosaceae, is a typical apomictic triploid plant. Pingyi Tiancha has high apomictic ability and is an important female parent in the apomictic dwarf rootstock breeding. Using the character of facultative apomixis in most apomictic plants of Malus, the tetraploid leaf-wrinkled and dwarf hybrids crossed by Pingyi Tiancha and Zha'ai Shandingzi (M. baccata) were obtained in the early study.The further research results showed that in most hybrids their apomictic ability declined significantly. Till now there are very little research and reports in molecular mechanism to reveal why apomixes rate declined significantly in the apomictic hybrids and in the reasons for this decline it is caused by some genes'controlling.
In this research, using the triploid Pingyi Tiancha and tetraploid hybrid strain33#as paint materials, we investigated the mechanism of apomixes at the molecular level through cloning the apomixis-related genes, analysing the gene expression differences in the reproductive process, constructing vector and making genetic transformation.The main results are as follows:
1.4homologous SERK genes'fragments were obtained by homologous clone from genome DNA of Pingyi Tiancha and hybrid strain33#, and the full-length of them were705bp,671bp,504bp,740bp and705bp,500bp,639bp,675bp, respectively.The homologous gene fragments had the similar structure of an intron, which had high homology with SERK homologous genes from other plants.
2. Using the published genome sequence in Malus, cDNA full-length sequences of SERK1and SERK4were isolated from Pingyi Tiancha and the hybrid strain33#, named MhSERK1, MhdSERK, MhSERK4and MhdSERK4. The cDNA full-length sequences of MhSERK1and MhdSERK1were1899bp and1881bp, which encoded632and626amino acids, respectively. After comparing the cDNA sequences between MhSERKl and MhdSERK1it was found that there was18bp difference at696-714bp, which might be related with apomictic ability.The length of MhSERK4and MhdSERK4were both1836bp and had a99.4%homology, encoding611amino acids, respectively. The amino acids sequence has the highest homology of more than90%with coconut, and the kinase structure area was highly conserved which had the typical LRR-RLKs structural character of SERK gene family.
3. Based on the obtained cDNA sequence, the full-length sequences of MhSERKl and MhdSERK1in DNA coding region were obtained by PCR. The full-length sequences in DNA coding region of MhSERK1and MhdSERKl were6886bp and6719bp, both with11exons and10introns. The distribution of introns in MhSERKl and MhdSERKl were similar with that in Longan (accession number:HM773391) and Papaya (accession number: EF661025).
4. SERKs expression was detected in different tissues, organs and flower's different developmental stages of triploid Pingyi Tiancha (3n) and tetraploid hybrid strain33#(4n) through Real-time quantitative PCR methodThe expression of SERK1, SERK1, SERK1and SERK4were mainly occurred in the reproductive tissues, and expression was higher in Pingyi Tiancha than in hybrid strain, indicating that SERK gene was related to apomixes. Expression analysis on flower's different developmental stages showed that SERK1expression was highest in the bud period of Pingyi Tiancha, then the full-blossom period, and it was lowest after flowering; while it was highest after flowering in hybrid strain which had lower expression in the bud period and the lowest expression in the full-blossom period. Expression of SERK1was the highest in the ovary of both Pingyi Tiancha and hybrid strain33#, in stamen was higher, In the petal, however, expression was the lowest in Pingyi Tiancha. Expression were occurred in the leaf, petal and pistil of hybrid strain33#, but it was nearly no expression. The highest expression of SERK2gene was occurred in the second day after flowering in Pingyi Tiancha, which was5times more than that in hybrids33#; and the highest expression was occurred in the third day after flowering in hybrid strain33#, which was2times bigger than that in Pingyi Tiancha. SERK3gene expression in Pingyi Tiancha reached the maximum in the second day after flowering and about4times larger than that in hybrid strain33#; the expression of SERK4gene was higher in1-5d in Pingyi Tiancha than in hybrid strain33#, respectively, which were inserted into agrobacterium strain EHA105successfully.
5. Plant expression vectors of MhSERKl and MhdSERK1as well as MhSERK4and MhdSERK4were constructed, named pBI121-MhSERK1, pBI121-MhdSERKl, pBI121-MhSERK4and pBI121-MhdSERK4, and transformed the SERKl gene into tobacco successfully.
6. Genetic transplant study was carried out based on Micro-Tom tomato and tobacco as materials by using agrobacterium infection method and constructed pBI121-MhSERKl and pBI121-MhdSERKl plant expression vectors,7and6tobacco resistant plants were obtained, and the PCR detection showed that transformation was successful.
本研究以苹果属三倍性平邑甜茶和四倍性杂种后代株系为试材,克隆无融合生殖相关SERK基因,分析无融合生殖基因在生殖过程中的表达差异,并通过构建植物表达载体及遗传转化的研究,以期从分子水平探讨苹果属无融合生殖的分子机理。主要研究结果如下：
1.采用同源克隆的方法分别从平邑甜茶和杂种后代33#株系基因组DNA中克隆得到4个SERK同源基因家族片段,其长度分别为705bp、671bp、504bp、740bp;705bp、500bp、639bp、675bp。它们的结构基本相同,都具有一个内含子,与其他植物的SERK同源基因具有较高的同源性。
2.利用已公布的苹果基因组序列从平邑甜茶和杂种后代33#株系中分离出了SERK1和SERK4基因的cDNA全长序列,分别将其命名为MhSERK1、MhdSERK1和MhSERK4、MhdSERK4。其中MhSERK1、MhdSERK1的cDNA长度为1899bp、1881bp,分别编码632、626个氨基酸。比对MhSERK1和MhdSERK1的cDNA序列在696-714bp处存在18bp的缺失差异,分析这段差异可能与其无融合生殖能力有关。MhSERK4、 MhdSERK4的两条片段长度均为1836bp,同源性达到99.40%,分别编码611个氨基酸。氨基酸序列与棕榈科植物椰子同源性最高,达到90%以上；氨基酸编码蛋白激酶结构域高度保守,具有SERK类家族典型的LRR-RLKs结构特点。
3.根据已获得的cDNA序列,设计引物扩增仅得到了MhSERK1和MhdSERK1的DNA编码区全长序列。MhSERK1和MhdSERK1DNA编码区序列全长分别为6886bp、6719bp,均有11个外显子,10个内含子,MhSERK1和MhdSERK1的内含子分布与龙眼(HM773391)和番木瓜(EF661025)等同源基因类似,都具有11个外显子和10个内含子结构。
4.利用实时定量RT-PCR的方法分别检测了SERK基因在三倍性平邑甜茶和四倍性杂种后代33#株系不同器官、组织及子房发育不同时期的表达情况o SERK1、SERK2、 SERK3、SERK4基因主要在生殖器官中表达,而且在生殖器官中的表达平邑甜茶明显高于杂种后代33#,说明SERK基因是与无融合生殖相关的基因；在不同花期子房的表达分析SERK1在平邑甜茶花蕾期(花前5天)子房中表达量最高,其次是盛花期,盛花期后(花后5天)的子房中表达量最低；而在杂种后代中盛花期后的表达量最高,其次是花蕾期,在盛花期表达量最低。SERK1无论在平邑甜茶还是在杂种后代33#的子房中都是表达量最高,其次是在雄蕊中；而在平邑甜茶花瓣中表达量最低,在杂种后代33#的叶片、花瓣和雌蕊中表达量都很低,几乎不表达。SERK2基因在平邑甜茶开花后第2d的子房中表达量最高,大约是杂种后代33#表达量的5倍；而在杂种后代33#开花后第3d的表达量最高,大约是平邑甜茶表达量的二倍。SERK3基因在平邑甜茶开花后第2d子房中的表达量最高,大约是杂种后代33#表达量的15倍；SERK4基因在平邑甜茶开花后第1d的表达量最高,而且在平邑甜茶花后1-5d的表达量均高于杂种后代33#株系。
5.构建了MhSERK1、MhdSERK1和MhSERK4、MhdSERK4植物表达载体,将其分别命名为pBI121-MhSERK1、pBI121-MhdSERK1和pBI121-MhSERK4、 pBI121-MhdSERK4,并成功导入农杆菌菌株EHA105。
6.通过农杆菌侵染法,利用构建好的pBI121-MhSERK1、pBI121-MhdSERKl植物表达载体,分别以Micro-Tom番茄和烟草为试材进行遗传转化研究,共获得7株和6株烟草抗性植株,通过PCR检测验证转化成功。
Pingyi Tiancha(Malus hupehensis Rehd. var. pingyiensis Jiang), a variety of crabapple (Malus hupehensis (Pamp.) Rehd.), which belongs to the Malus of Rosaceae, is a typical apomictic triploid plant. Pingyi Tiancha has high apomictic ability and is an important female parent in the apomictic dwarf rootstock breeding. Using the character of facultative apomixis in most apomictic plants of Malus, the tetraploid leaf-wrinkled and dwarf hybrids crossed by Pingyi Tiancha and Zha'ai Shandingzi (M. baccata) were obtained in the early study.The further research results showed that in most hybrids their apomictic ability declined significantly. Till now there are very little research and reports in molecular mechanism to reveal why apomixes rate declined significantly in the apomictic hybrids and in the reasons for this decline it is caused by some genes'controlling.
In this research, using the triploid Pingyi Tiancha and tetraploid hybrid strain33#as paint materials, we investigated the mechanism of apomixes at the molecular level through cloning the apomixis-related genes, analysing the gene expression differences in the reproductive process, constructing vector and making genetic transformation.The main results are as follows:
1.4homologous SERK genes'fragments were obtained by homologous clone from genome DNA of Pingyi Tiancha and hybrid strain33#, and the full-length of them were705bp,671bp,504bp,740bp and705bp,500bp,639bp,675bp, respectively.The homologous gene fragments had the similar structure of an intron, which had high homology with SERK homologous genes from other plants.
2. Using the published genome sequence in Malus, cDNA full-length sequences of SERK1and SERK4were isolated from Pingyi Tiancha and the hybrid strain33#, named MhSERK1, MhdSERK, MhSERK4and MhdSERK4. The cDNA full-length sequences of MhSERK1and MhdSERK1were1899bp and1881bp, which encoded632and626amino acids, respectively. After comparing the cDNA sequences between MhSERKl and MhdSERK1it was found that there was18bp difference at696-714bp, which might be related with apomictic ability.The length of MhSERK4and MhdSERK4were both1836bp and had a99.4%homology, encoding611amino acids, respectively. The amino acids sequence has the highest homology of more than90%with coconut, and the kinase structure area was highly conserved which had the typical LRR-RLKs structural character of SERK gene family.
3. Based on the obtained cDNA sequence, the full-length sequences of MhSERKl and MhdSERK1in DNA coding region were obtained by PCR. The full-length sequences in DNA coding region of MhSERK1and MhdSERKl were6886bp and6719bp, both with11exons and10introns. The distribution of introns in MhSERKl and MhdSERKl were similar with that in Longan (accession number:HM773391) and Papaya (accession number: EF661025).
4. SERKs expression was detected in different tissues, organs and flower's different developmental stages of triploid Pingyi Tiancha (3n) and tetraploid hybrid strain33#(4n) through Real-time quantitative PCR methodThe expression of SERK1, SERK1, SERK1and SERK4were mainly occurred in the reproductive tissues, and expression was higher in Pingyi Tiancha than in hybrid strain, indicating that SERK gene was related to apomixes. Expression analysis on flower's different developmental stages showed that SERK1expression was highest in the bud period of Pingyi Tiancha, then the full-blossom period, and it was lowest after flowering; while it was highest after flowering in hybrid strain which had lower expression in the bud period and the lowest expression in the full-blossom period. Expression of SERK1was the highest in the ovary of both Pingyi Tiancha and hybrid strain33#, in stamen was higher, In the petal, however, expression was the lowest in Pingyi Tiancha. Expression were occurred in the leaf, petal and pistil of hybrid strain33#, but it was nearly no expression. The highest expression of SERK2gene was occurred in the second day after flowering in Pingyi Tiancha, which was5times more than that in hybrids33#; and the highest expression was occurred in the third day after flowering in hybrid strain33#, which was2times bigger than that in Pingyi Tiancha. SERK3gene expression in Pingyi Tiancha reached the maximum in the second day after flowering and about4times larger than that in hybrid strain33#; the expression of SERK4gene was higher in1-5d in Pingyi Tiancha than in hybrid strain33#, respectively, which were inserted into agrobacterium strain EHA105successfully.
5. Plant expression vectors of MhSERKl and MhdSERK1as well as MhSERK4and MhdSERK4were constructed, named pBI121-MhSERK1, pBI121-MhdSERKl, pBI121-MhSERK4and pBI121-MhdSERK4, and transformed the SERKl gene into tobacco successfully.
6. Genetic transplant study was carried out based on Micro-Tom tomato and tobacco as materials by using agrobacterium infection method and constructed pBI121-MhSERKl and pBI121-MhdSERKl plant expression vectors,7and6tobacco resistant plants were obtained, and the PCR detection showed that transformation was successful.
引文
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