一个亚洲棉不育矮秆突变体的分离鉴定及相关基因克隆
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摘要
株高是棉花的一个重要性状,在棉花栽培中矮化对于塑造株形、提高产量具有重要作用,因此矮化育种一直是棉花育种的一个热点。本试验通过对矮秆突变体的遗传、解剖结构、生理生化方面的研究,旨在阐明矮杆表型的分子机制。本试验将来源于亚洲棉(Gossypium arboreum L.)品种金华中棉经60Co γ-射线照射后,分离、鉴定出一个新不育矮秆突变体,遗传分析结果显示不育矮秆表型可能是由一对隐性等位基因控制,因此,我们建议将不育矮秆突变体命名为sda。sda植株矮化是节间长度缩短与节间数减少两者共同作用的结果,扫描电镜观察结果表明节间长度与节间细胞数目有关。维管形成层的活动明显,次生组织比例大；次生木质部细胞木质化程度高；后生木质部导管数增加,且导管排列散乱；位于次生韧皮部与皮层之间的韧皮纤维十分明显。另外与野生型(WT)相比,sda植株吲哚-3-乙酸(IAA)与脱落酸(ABA)含量较低。而且,突变体叶片内的叶绿素含量与净光合速率明显降低。但是ABA生物合成或信号转导与sda表型形成可能有关。通过半定量RT-PCR分析检测到13条差异表达的ESTs,不育矮秆突变体表达水平降低,野生型表达水平增加。结合9个潜在的激素生物合成基因在IAA、ABA、多胺(PA)和茉莉酸(JA)合成中的作用对本研究结果进行了讨论。
为确定吲哚乙酸生物合成基因色氨酸脱羧酶与腈水解酶在IAA变化中的作用,利用反转录聚合酶链式反应(RT-PCR)及快速扩增cDNA末端(RACE)从亚洲棉叶片中克隆到这两个基因。棉花TDC(GaTDC)全长包含148bp的5’非翻译区,一个含有1494bp的开放阅读框(ORF),编码一条含有497个氨基酸的多肽,该多肽的分子量估计为54.898KD,和185bp的3’非翻译区。GaTDC编码的氨基酸序列与其它生物体TDC具有高度的同源性。通过实时定量PCR (qRT-PCR)试验检测到sda突变体叶片中GaTDC的mRNA表达水平低下。相对于野生型,突变体GaTDC表达下调。GaNIT cDNA序列全长为1502bp,它包括150bp的5’非翻译区,一个1053bp的开放阅读框,和299bp的3’非翻译区。编码的GaNIT预测为350氨基酸长,分子量计算值为37.943KD,等电点是5.19。发现在氨基酸水平棉花腈水解酶与已知的腈水解酶序列之间高度同源。利用qRT-PCR分析检测到它的mRNA转录本在突变体叶片中下调表达。本研究结果表明sda植株吲哚-3-乙酸(IAA)的含量低于野生型植株的吲哚-3-乙酸(IAA),突变体内GaTDC表达和GaNIT表达减少。结果对探讨GaTDC和GaNIT在棉花IAA生物合成中的功能是有益的。
根据编码玉米黄质环氧化酶(ZEP)的EST片段序列,利用RACE策略快速克隆到一个与黄化相关的新基因(EAG)全长cDNA,它编码包含FAD结合结构域和FHA结构域的蛋白质,用GaZEP表示。实时定量PCR结果显示突变体内GaZEP表达下调。对玉米黄质环氧化酶在叶黄素循环和ABA前体合成中的作用相关结果进行了讨论。
采用PCR扩增与锚定PCR技术从亚洲棉中克隆了精氨酸脱羧酶(ADC)基因。棉花ADC(GaADC)全长cDNA包括474bp的5’非翻译区(UTR),一个2181bp的ORF,编码726个氨基酸的多肽,估计该多肽的分子量为78.078KD,和382bp的3’非翻译区。GaADC编码的氨基酸序列与其它生物ADC具有高度的同源性。应用实时定量PCR (qRT-PCR)分析评估不同材料内GaADC的mRNA表达,检测到sda叶片中GaADC的mRNA表达水平低下。60Co γ射线照射后sda内GaADC的表达下调,结果表明GaADC可能为涉及植物衰老的一种蛋白质。
为了充分证明核DNA出现的辐射损伤,本试验对60Co γ射线的特性和其辐射强度与种子损伤之间的关系做了进一步的研究。高剂量辐射对棉花、尤其是棉花基因组的直接影响没有得到充分的证明。将棉花种子暴露于剂量高达300Gy的60Co γ,辐射下,通过对DNA进行突变与缺失分析,采用SSR引物评估出现诱导的可能性,进行DNA测序分析鉴定诱导的点突变。序列分析表明γ射线诱导sda突变主要包括部分缺失与点突变。
Plant height is an important cotton trait, and dwarf plants are most desirable in cotton cultivation. Therefore, advances in dwarf breeding programs remain a focus in cotton propagation. The present study served to elucidate the molecular mechanisms of the dwarf stem phenotype. A sterile-dwarf mutant from strain Jinhuazhongmian of Gossypium arboreum L. was irradiated by60Co γ-ray, and subsequently isolated and characterized. Results indicated that the sterile-dwarf phenotype was conferred by one pair of recessive alleles. A sterile-dwarf recombinant was detected and termed sd-a. The combined action of the internodal length reduction and intermodal number decrease resulted in sd(?) plant dwarf. Scanning electron microscope (SEM) observation showed that internodal length was in relation to cell number of internode. The activity of vascular cambium was significant, of which secondary tissues accounted for a large proportion. The degree of cell lignifications in secondary xylem was higher. The vessel number in metaxylem increased. Moreover, vessels arrayed disorderly. Phloem fiber presented between secondary phloem and cortex significantly. The sd(?) plants contained lower levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) compared with wild-type (WT) plants. Furthermore, the contents of chlorophyll and net photosynthetic rate in leaves of the mutant obviously decreased. However, it is plausible ABA biosynthesis or signaling was involved in the formation of sd(?) phenotypes. Semi-quantitative RT-PCR analysis detected13differentially expressed ESTs, with the sterile-dwarf mutant exhibiting decreased and the WT increased levels of expression. The results of this study were discussed in light of the role of nine potential hormone biosynthetic genes in the synthesis of IAA, ABA, PA, and JA.
To determine the role of the indole acetic acid biosynthetic genes TDC and NIT in IAA change, we cloned these two genes from Gossypium arboreum leaves by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length cDNA of asian cotton TDC(GaTDC) contained a5'untranslated region (UTR) of148bp, an ORF (open reading frame) of1494bp encoding a polypeptide of497amino acids with an estimated molecular mass of54.898kDa and a3'UTR of185bp. The predicted amino acid sequence of GaTDC shared high identity with that of TDC in other organisms. Lower-level mRNA expression of GaTDC was detected in the leaves of sd(?) mutant by Real-Time quantitative PCR (qRT-PCR) assay. Relative to wild type, the expression of GaTDC in the mutant was down regulated. The full length of the GaNIT cDNA sequence was1502bp, including a150bp5'UTR, a1053bp open reading frame, and a299bp3'UTR. The putative GaNIT is predicted to be350amino acids long, with a calculated molecular mass of37.943kDa and an isoelectric point of5.19. High homologies were found at the amino acid level between G. arboreum nitrilase and the sequences of known nitrilases. Its mRNA transcript was found to be down-regulated expression in leaves of the mutant examined by qRT-PCR analysis. Here we showed that sd(?) plants contained lower levels of indole-3-acetic acid (IAA) than wild-type plants. Here, we found that GaTDC expression and GaNIT expression are decreased in mutant. These results will help to explore the function of both GaTDC and GaNIT in cotton IAA synthesis.
According to the sequence of an EST fragment encoding a zeaxanthin epoxidase (ZEP), a novel etiolation-associated gene (EAG) full-length cDNA encoding a FAD binding domain and a FHA domain-containing protein, denoted GaZEP, was rapidly cloned using a strategy of RACE. It was demonstrated by real-time quantitative PCR that the expression of GaZEP in the mutant was down regulated. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.
The techniques of PCR amplification cloning and anchored PCR were used to clone the arginine decarboxylase (ADC) gene from asian cotton (Gossypium arboreum L.). The full length cDNA of Gossypium arboreum ADC (GaADC) contained a5'untranslated region (UTR) of474bp, an ORF (open reading frame) of2181bp encoding a polypeptide of726amino acids with an estimated molecular mass of78.078kDa and a3'UTR of382bp. The predicted amino acid sequence of GaADC shared high identity with ADC in other organisms. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of GaADC in different lines. Lower-level mRNA expression of GaADC was detected in the leaves of sd(?). The expression of GaADC in the sd(?) was down regulated after irradiated by60Co y-ray. The results indicated that GaADC might be a protein involved in plant senescence.
To well document radiation damage incurred by nuclear DNA, interest is increasing in the properties of60Co γ-ray and their ability to induce damage in seeds exposed to radiation. Direct high-dose radiation effects on the cotton and more particularly the cotton genome are less well understood. In this study cotton seeds were exposed to y-radiation doses as high as300Gy from60Co. Mutation and deletion analysis was performed on DNA. SSR Primers were employed to assess its possible induction. DNA sequencing analysis was conducted to identify induced point mutations. Sequence analysis revealed that y-ray-induced mutation in sd(?) included mainly partial deletion and point mutation.
为确定吲哚乙酸生物合成基因色氨酸脱羧酶与腈水解酶在IAA变化中的作用,利用反转录聚合酶链式反应(RT-PCR)及快速扩增cDNA末端(RACE)从亚洲棉叶片中克隆到这两个基因。棉花TDC(GaTDC)全长包含148bp的5’非翻译区,一个含有1494bp的开放阅读框(ORF),编码一条含有497个氨基酸的多肽,该多肽的分子量估计为54.898KD,和185bp的3’非翻译区。GaTDC编码的氨基酸序列与其它生物体TDC具有高度的同源性。通过实时定量PCR (qRT-PCR)试验检测到sda突变体叶片中GaTDC的mRNA表达水平低下。相对于野生型,突变体GaTDC表达下调。GaNIT cDNA序列全长为1502bp,它包括150bp的5’非翻译区,一个1053bp的开放阅读框,和299bp的3’非翻译区。编码的GaNIT预测为350氨基酸长,分子量计算值为37.943KD,等电点是5.19。发现在氨基酸水平棉花腈水解酶与已知的腈水解酶序列之间高度同源。利用qRT-PCR分析检测到它的mRNA转录本在突变体叶片中下调表达。本研究结果表明sda植株吲哚-3-乙酸(IAA)的含量低于野生型植株的吲哚-3-乙酸(IAA),突变体内GaTDC表达和GaNIT表达减少。结果对探讨GaTDC和GaNIT在棉花IAA生物合成中的功能是有益的。
根据编码玉米黄质环氧化酶(ZEP)的EST片段序列,利用RACE策略快速克隆到一个与黄化相关的新基因(EAG)全长cDNA,它编码包含FAD结合结构域和FHA结构域的蛋白质,用GaZEP表示。实时定量PCR结果显示突变体内GaZEP表达下调。对玉米黄质环氧化酶在叶黄素循环和ABA前体合成中的作用相关结果进行了讨论。
采用PCR扩增与锚定PCR技术从亚洲棉中克隆了精氨酸脱羧酶(ADC)基因。棉花ADC(GaADC)全长cDNA包括474bp的5’非翻译区(UTR),一个2181bp的ORF,编码726个氨基酸的多肽,估计该多肽的分子量为78.078KD,和382bp的3’非翻译区。GaADC编码的氨基酸序列与其它生物ADC具有高度的同源性。应用实时定量PCR (qRT-PCR)分析评估不同材料内GaADC的mRNA表达,检测到sda叶片中GaADC的mRNA表达水平低下。60Co γ射线照射后sda内GaADC的表达下调,结果表明GaADC可能为涉及植物衰老的一种蛋白质。
为了充分证明核DNA出现的辐射损伤,本试验对60Co γ射线的特性和其辐射强度与种子损伤之间的关系做了进一步的研究。高剂量辐射对棉花、尤其是棉花基因组的直接影响没有得到充分的证明。将棉花种子暴露于剂量高达300Gy的60Co γ,辐射下,通过对DNA进行突变与缺失分析,采用SSR引物评估出现诱导的可能性,进行DNA测序分析鉴定诱导的点突变。序列分析表明γ射线诱导sda突变主要包括部分缺失与点突变。
Plant height is an important cotton trait, and dwarf plants are most desirable in cotton cultivation. Therefore, advances in dwarf breeding programs remain a focus in cotton propagation. The present study served to elucidate the molecular mechanisms of the dwarf stem phenotype. A sterile-dwarf mutant from strain Jinhuazhongmian of Gossypium arboreum L. was irradiated by60Co γ-ray, and subsequently isolated and characterized. Results indicated that the sterile-dwarf phenotype was conferred by one pair of recessive alleles. A sterile-dwarf recombinant was detected and termed sd-a. The combined action of the internodal length reduction and intermodal number decrease resulted in sd(?) plant dwarf. Scanning electron microscope (SEM) observation showed that internodal length was in relation to cell number of internode. The activity of vascular cambium was significant, of which secondary tissues accounted for a large proportion. The degree of cell lignifications in secondary xylem was higher. The vessel number in metaxylem increased. Moreover, vessels arrayed disorderly. Phloem fiber presented between secondary phloem and cortex significantly. The sd(?) plants contained lower levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) compared with wild-type (WT) plants. Furthermore, the contents of chlorophyll and net photosynthetic rate in leaves of the mutant obviously decreased. However, it is plausible ABA biosynthesis or signaling was involved in the formation of sd(?) phenotypes. Semi-quantitative RT-PCR analysis detected13differentially expressed ESTs, with the sterile-dwarf mutant exhibiting decreased and the WT increased levels of expression. The results of this study were discussed in light of the role of nine potential hormone biosynthetic genes in the synthesis of IAA, ABA, PA, and JA.
To determine the role of the indole acetic acid biosynthetic genes TDC and NIT in IAA change, we cloned these two genes from Gossypium arboreum leaves by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length cDNA of asian cotton TDC(GaTDC) contained a5'untranslated region (UTR) of148bp, an ORF (open reading frame) of1494bp encoding a polypeptide of497amino acids with an estimated molecular mass of54.898kDa and a3'UTR of185bp. The predicted amino acid sequence of GaTDC shared high identity with that of TDC in other organisms. Lower-level mRNA expression of GaTDC was detected in the leaves of sd(?) mutant by Real-Time quantitative PCR (qRT-PCR) assay. Relative to wild type, the expression of GaTDC in the mutant was down regulated. The full length of the GaNIT cDNA sequence was1502bp, including a150bp5'UTR, a1053bp open reading frame, and a299bp3'UTR. The putative GaNIT is predicted to be350amino acids long, with a calculated molecular mass of37.943kDa and an isoelectric point of5.19. High homologies were found at the amino acid level between G. arboreum nitrilase and the sequences of known nitrilases. Its mRNA transcript was found to be down-regulated expression in leaves of the mutant examined by qRT-PCR analysis. Here we showed that sd(?) plants contained lower levels of indole-3-acetic acid (IAA) than wild-type plants. Here, we found that GaTDC expression and GaNIT expression are decreased in mutant. These results will help to explore the function of both GaTDC and GaNIT in cotton IAA synthesis.
According to the sequence of an EST fragment encoding a zeaxanthin epoxidase (ZEP), a novel etiolation-associated gene (EAG) full-length cDNA encoding a FAD binding domain and a FHA domain-containing protein, denoted GaZEP, was rapidly cloned using a strategy of RACE. It was demonstrated by real-time quantitative PCR that the expression of GaZEP in the mutant was down regulated. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.
The techniques of PCR amplification cloning and anchored PCR were used to clone the arginine decarboxylase (ADC) gene from asian cotton (Gossypium arboreum L.). The full length cDNA of Gossypium arboreum ADC (GaADC) contained a5'untranslated region (UTR) of474bp, an ORF (open reading frame) of2181bp encoding a polypeptide of726amino acids with an estimated molecular mass of78.078kDa and a3'UTR of382bp. The predicted amino acid sequence of GaADC shared high identity with ADC in other organisms. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of GaADC in different lines. Lower-level mRNA expression of GaADC was detected in the leaves of sd(?). The expression of GaADC in the sd(?) was down regulated after irradiated by60Co y-ray. The results indicated that GaADC might be a protein involved in plant senescence.
To well document radiation damage incurred by nuclear DNA, interest is increasing in the properties of60Co γ-ray and their ability to induce damage in seeds exposed to radiation. Direct high-dose radiation effects on the cotton and more particularly the cotton genome are less well understood. In this study cotton seeds were exposed to y-radiation doses as high as300Gy from60Co. Mutation and deletion analysis was performed on DNA. SSR Primers were employed to assess its possible induction. DNA sequencing analysis was conducted to identify induced point mutations. Sequence analysis revealed that y-ray-induced mutation in sd(?) included mainly partial deletion and point mutation.
引文
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