水稻vdac、ant及HL-sp1基因的表达研究
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摘要
植物细胞质雄性不育(Cytoplasmic male sterility, CMS)是普遍存在于高等植物中的一种生物学现象,在遗传育种中有着重要的应用价值。大多数植物中,CMS的产生与线粒体基因组功能异常相关,如水稻红莲型CMS(HL-CMS)与其线粒体中的两个特有基因HL-sp1和orfH79有关。线粒体的功能也受核编码的蛋白的调控,如电压依赖性阴离子通道(Voltage-dependent anion channel, VDAC)蛋白参与包括NADH和ATP在内的离子和小分子跨线粒体外膜运输,在线粒体新陈代谢和离子信号传导中起重要作用;腺苷酸转位酶(Adenine Nucleotide Translocase, ANT)介导了线粒体内膜上ATP与ADP交换转运,ANT介导线粒体氧化磷酸化在细胞能量代谢中发挥重要作用。本研究探讨了HL-sp1、vdac和ant基因在水稻HL-CMS不育系中的表达,得到了如下主要结果:
1.对水稻日本晴基因组中已预测得到的8个vdac基因及其表达产物进行了生物信息学对比分析。以水稻HL-CMS不育系YTA为材料,采用实时荧光定量PCR(real-time PCR)技术,比较分析了这8个vdac基因在YTA不同组织和不同发育时期的表达特性。结果表明,osvdac1和osvdac4在YTA各发育时期均有较高表达;在非生物胁迫(干旱、NaCl和甘露醇胁迫)及胁迫去除后8个vdac基因表达有明显差异。在此基础上,利用SMARTer RACE技术对osvdac4基因5' UTR和3' UTR序列进行分析,已得到该基因完整的3' UTR序列。
2.利用real-time PCR技术比较分析了3个ant基因在水稻HL-CMS不育系YTA不同生长与发育时期的不同组织中的表达特性。结果表明,osant1和osant2在YTA营养生长的根、茎、叶中的表达量均较高,而在生殖生长的不同时期小穗或花药中的表达量显著降低。osant3的表达模式较前两者明显不同,在YTA全生育期均为低水平表达。
3.利用SMARTer RACE技术研究orf216基因完整的5' UTR和3' UTR序列,已获得该基因完整的3' UTR序列,5' UTR分析发现orf216基因与其上游基因共转录。
Cytoplasmic male sterility (CMS) is a very common phenomenon in plant kingdom which plays an important role in genetic breeding. In most plants, CMS is in relation to mitochondrial genomic dysfunction. For instances, the mitochondria-associated genes, HL-sp1 and orfH79, are involved in the Honglian CMS (HL-CMS) in rice. The function of mitochondria is also regulated by nuclear genes. The voltage-dependent anion channel protein (VDAC), which across the mitochondrial outer membrane, coded by nuclear genome is involved in the transport of metabolites, including NADH and ATP, and plays an important role in mitochondrial metabolism and ion signaling. Adenine nucleotide translocase (ANT) coded by nuclear genome is an inner membrane transporter that facilitates the exchange of ATP and ADP and is associated in cellular energy metabolism by influencing mitochondrial oxidative phosphorylation. In this study, the expressions of HL-sp1, vdac and ant in rice were carried out. The results are as follow:
1. The bioinformatics analysis on eight vdac genes, previously predicted based on Nipponbare genome in rice, and their expression products was first carried out. The expression models of eight vdac genes in the different tissues of HL-CMS sterile line Yuetai A (YTA) at different growth and development stages were analyzed by method of real-time PCR. The results showed that the expressions of osvdac1 and osvdac4 were much higher among the eight vdac genes during the whole growth stages of YTA. When being treated with abiotic stress of drought, 200 mmol/L NaCl and 360 mmol/L mannitol, the expression patterns of eight vdac genes in different tissues of YTA were greatly changed during the periods of treatment and stress recovery. And one of vdac genes, osvdac4, was then selected, and its 5'-UTR and 3'-UTR sequences was analyzed by method of SMARTer RACE. According to sequencing data, the complete 3'-UTR sequence of osvdac4 gene was confirmed.
2. The expression patterns of three ant genes in the different tissues of YTA at different growth and development stages were analyzed by method of real-time PCR. The results indicated that the expression of osant1 and osant2 was higher at vegetative stages of YTA, but lower during reproductive stage. Osant3 was weakly expressed in most tissues of YTA at all growth and developmental stages.
3. SMARTer RACE was used to amplify the complete 5'-UTR and 3'-UTR sequences of orf216 gene. According to sequencing data of amplified cDNA, the complete 3'-UTR sequence of orf216 gene and its co-transcription with its upstream genes was confirmed.
1.对水稻日本晴基因组中已预测得到的8个vdac基因及其表达产物进行了生物信息学对比分析。以水稻HL-CMS不育系YTA为材料,采用实时荧光定量PCR(real-time PCR)技术,比较分析了这8个vdac基因在YTA不同组织和不同发育时期的表达特性。结果表明,osvdac1和osvdac4在YTA各发育时期均有较高表达;在非生物胁迫(干旱、NaCl和甘露醇胁迫)及胁迫去除后8个vdac基因表达有明显差异。在此基础上,利用SMARTer RACE技术对osvdac4基因5' UTR和3' UTR序列进行分析,已得到该基因完整的3' UTR序列。
2.利用real-time PCR技术比较分析了3个ant基因在水稻HL-CMS不育系YTA不同生长与发育时期的不同组织中的表达特性。结果表明,osant1和osant2在YTA营养生长的根、茎、叶中的表达量均较高,而在生殖生长的不同时期小穗或花药中的表达量显著降低。osant3的表达模式较前两者明显不同,在YTA全生育期均为低水平表达。
3.利用SMARTer RACE技术研究orf216基因完整的5' UTR和3' UTR序列,已获得该基因完整的3' UTR序列,5' UTR分析发现orf216基因与其上游基因共转录。
Cytoplasmic male sterility (CMS) is a very common phenomenon in plant kingdom which plays an important role in genetic breeding. In most plants, CMS is in relation to mitochondrial genomic dysfunction. For instances, the mitochondria-associated genes, HL-sp1 and orfH79, are involved in the Honglian CMS (HL-CMS) in rice. The function of mitochondria is also regulated by nuclear genes. The voltage-dependent anion channel protein (VDAC), which across the mitochondrial outer membrane, coded by nuclear genome is involved in the transport of metabolites, including NADH and ATP, and plays an important role in mitochondrial metabolism and ion signaling. Adenine nucleotide translocase (ANT) coded by nuclear genome is an inner membrane transporter that facilitates the exchange of ATP and ADP and is associated in cellular energy metabolism by influencing mitochondrial oxidative phosphorylation. In this study, the expressions of HL-sp1, vdac and ant in rice were carried out. The results are as follow:
1. The bioinformatics analysis on eight vdac genes, previously predicted based on Nipponbare genome in rice, and their expression products was first carried out. The expression models of eight vdac genes in the different tissues of HL-CMS sterile line Yuetai A (YTA) at different growth and development stages were analyzed by method of real-time PCR. The results showed that the expressions of osvdac1 and osvdac4 were much higher among the eight vdac genes during the whole growth stages of YTA. When being treated with abiotic stress of drought, 200 mmol/L NaCl and 360 mmol/L mannitol, the expression patterns of eight vdac genes in different tissues of YTA were greatly changed during the periods of treatment and stress recovery. And one of vdac genes, osvdac4, was then selected, and its 5'-UTR and 3'-UTR sequences was analyzed by method of SMARTer RACE. According to sequencing data, the complete 3'-UTR sequence of osvdac4 gene was confirmed.
2. The expression patterns of three ant genes in the different tissues of YTA at different growth and development stages were analyzed by method of real-time PCR. The results indicated that the expression of osant1 and osant2 was higher at vegetative stages of YTA, but lower during reproductive stage. Osant3 was weakly expressed in most tissues of YTA at all growth and developmental stages.
3. SMARTer RACE was used to amplify the complete 5'-UTR and 3'-UTR sequences of orf216 gene. According to sequencing data of amplified cDNA, the complete 3'-UTR sequence of orf216 gene and its co-transcription with its upstream genes was confirmed.
引文
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