棉纤维相关基因时空表达与纤维品质关联分析及β-1，4-葡糖苷酶基因的转基因功能验证
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摘要
几千个基因在棉纤维发育过程中起作用,相关基因的分离鉴定以及功能研究起步较晚,但进展很快。本试验从已经报道的纤维发育相关基因中选出10个在棉纤维发育中优势表达的基因,以14个纤维品质具有差异的棉花品种(或品系)为研究材料,利用实时荧光定量PCR技术检测10个基因在不同纤维发育时期的相对表达量,以期研究这些基因的表达水平与纤维品质各指标的相关性。综合分析各基因在不同棉花品种(或品系)中于0DPA、5DPA、10DPA、15DPA、20DPA和23DPA的表达谱发现:GhExp1基因、GhCIPK1基因、GhPL基因为纤维伸长期优势表达基因;GhRacA和GhRacB在纤维伸长的前期及次生壁加厚期高表达;GhACT1在伸长期扣次生壁加厚期优势表达;纤维素合酶基因(GhCelA1和GhCelA3)在纤维伸长的后期至次生壁加厚期优势表达;蔗糖合酶GhSus1基因在纤维伸长早期和次生壁加厚期优势表达,GhSusA1的优势表达主要集中在0-5DPA这一时期。大部分基因在低表达时其表达高低与纤维品质显著相关,而在表达量高峰期其表达高低与纤维品质没有相关性。GhRacA基因的表达是个例外,GhRacA在23DPA高表达且表达量与纤维品质显著正相关。GhExp1基因在20DPA的表达水平与纤维比强度和整齐度呈显著负相关与伸长率呈极显著正相关;GhPL基因在23DPA的表达水平与纤维长度呈显著负相关;GhRacA基因在5DPA和23DPA的表达水平都与纤维的伸长率呈极显著的正相关;GhRacB基因在10DPA的表达水平与纤维长度和整齐度呈显著负相关;GhCelA1基因在5DPA的表达水平与纤维长度呈显著正相关,与马克隆值呈显著负相关,在10DPA的表达水平与马克隆值呈显著正相关,与伸长率达到极显著正相关,与比强度呈显著负相关,与纤维长度和整齐度呈极显著负相关;GhCIPK1、GhACT1、蔗糖合酶基因(GhSus1和GhSusA1)和GhCelA3 5个基因在14个棉花品种(或品系)纤维发育的各时期与纤维品质的各指标无显著相关性。
为了研究棉花内源β-1,4-葡糖苷酶基因(GhBG)在棉纤维发育中的可能功能,本试验构建了由纤维特异启动子E6驱动的GhBG基因植物表达载体,通过农杆菌介导法将GhBG基因转化泗棉3号,对再生植株进行标记基因和目的基因的PCR检测,来自6个克隆的56个单株是阳性植株,嫁接成活48株。部分植株的Southern杂交表明基因已导入棉花基因组中。获得6个愈伤系的17个单株的种子,对这些株系后代进行目的基因PCR检测和卡那霉素检测。目的基因的分离比例都符合孟德尔一对基因的分离比例(3:1),和卡那霉素检测结果一致;选育出了1个转GhBG基因的T_3纯合株系(504-5)。取株系504-5和对照开花当天(0DPA),开花后5 DPA、10 DPA、15 DPA、20DPA和23DPA的胚珠或纤维,提取其RNA,对GhBG基因进行荧光实时定量PCR分析,结果表明,GhBG基因在株系504-5中5DPA、10DPA和20DPA这三个时期表达量显著高于对照。对T_1和T_2纤维素含量测定结果发现,转基因植株和受体纤维素含量无差异;T_1和T_2代株系棉纤维样品进行HVI900(ICC标准)品质检测,对纯合株系的分析表明:转基因棉花纤维长度和比强度较对照相比有明显降低,而伸长率显著增加。由此推测,GhBG基因的过量表达对纤维长度和比强度有影响。
Thousands of genes play role during fiber development,though the identification and function research of these genes was initiated recently,the progress was very quick.In this paper,we selected 10 preferentially expressed genes for fiber development which had reported previously and 14 cotton varieties(or strains) with significantly different fiber quality.Using Q-PCR technique to evaluate the relative expression value of 10 tested genes in different fiber developmental stages in 14 cotton varieties(or strains),in order to study the relationship between temporal and spatial expression of these genes and fiber quality traits.The analysis of expression profile of these genes in different cotton varieties(or strains) indicate:GhExp1,GhPL and GhCIPK1 gene preferentially express during fiber elongation;GhRacA and GhRacB gene have high expression level in earlier stage of fiber elongation and the thickening period of secondary cell wall;The predominant expression periods of GhACT1 are fiber elongation and the secondary cell wall thickening period;Two cellulose synthase genes(GhCelA1 and GhCelA3) preferentially express during late stage of fiber elongation and the thickening period of secondary cell wall;GhSus1 have high expression level during thickening period of secondary cell wall and fiber elongation stage, and GhSusA1 gene preferentially express during fiber initiation stage and the earlier stage of fiber elongation.For most genes,the expression value in low expression level period has significant correlation with fiber quality,while no significant correlation is detected when the expression of these genes at the peak except GhRacA gene.The expression level of GhExp1 in 20DPA fiber has a significant negative correlation with fiber strength and uniformity and it also has a significant positive correlation with fiber elongation;the expression level of GhPL gene in 23DPA has a significant negative correlation with fiber length;the expression level of GhRacA gene in 5DPA and 23DPA both have a high significant positive correlation with elongation;the expression level of GhRacB gene in 10DPA has a significant negative correlation with length;the expression level of GhCelA1 gene in 5DPA has a significant positive correlation with length and a significant negative correlation with Mic value;the expression level of GhCelA1 gene in 10DPA has a significant negative correlation with fiber length and a significant positive correlation with micronaire value and a high significant correlation with elongation;the expression level of GhCIPK1,GhACT1,GhSus1,GhSusA1and GhCelA3 gene have no significant correlation with fiber quality traits.
To characterize the possible function ofβ-1,4-glucosidase(GhBG) in cotton fiber,in this study,the sense coding sequence of the GhBG gene was fused with fiber-specific E6 promoter to build the constructs of sense gene,and with which to transform into Simian3 via Agrobacterium-mediated transformation.PCR amplification of the regenerated plants with primers of marker gene and target gene indicated that 56 plants from 6 clones were positive,of which 48 were survived by grafting.Southern blot analysis of the transgenic cotton plants showed that the introduction of GhBG gene into cotton genome was successful.Genetic Analysis of 17 T_1 and T_2 regenerated plants from 6 different transgenic lines by PCR analysis and Kanamycin-resistance assay indicated that the segregation ratio of GhBG gene in 6 transgenic lines were inherited in a classical Mendelian manner(3:1) and one pure line(504-5) was screened.For analyzing the expression level of this pure line, 0DPA,5DPA,10DPA,15DPA,20DPA and 23DPA ovule or fiber RNA of pure line was extracted,with Simian3 as control.The analysis of Q-PCR results showed that the expression level of GhBG gene in 5DPA,10DPA and 20DPA ovule or fiber of pure line was higher than that of control and the differences were significant.The result of mature celullose content test of T_1 and T_2 indicted that there was no significant difference between transgenic lines and control.Analysis of fiber quality traits showed that the fiber length and strength in pure line decreased and fiber elongation percentage increased.These results demonstrate that the overexpression of GhBG gene have effect to fiber length and strength.
为了研究棉花内源β-1,4-葡糖苷酶基因(GhBG)在棉纤维发育中的可能功能,本试验构建了由纤维特异启动子E6驱动的GhBG基因植物表达载体,通过农杆菌介导法将GhBG基因转化泗棉3号,对再生植株进行标记基因和目的基因的PCR检测,来自6个克隆的56个单株是阳性植株,嫁接成活48株。部分植株的Southern杂交表明基因已导入棉花基因组中。获得6个愈伤系的17个单株的种子,对这些株系后代进行目的基因PCR检测和卡那霉素检测。目的基因的分离比例都符合孟德尔一对基因的分离比例(3:1),和卡那霉素检测结果一致;选育出了1个转GhBG基因的T_3纯合株系(504-5)。取株系504-5和对照开花当天(0DPA),开花后5 DPA、10 DPA、15 DPA、20DPA和23DPA的胚珠或纤维,提取其RNA,对GhBG基因进行荧光实时定量PCR分析,结果表明,GhBG基因在株系504-5中5DPA、10DPA和20DPA这三个时期表达量显著高于对照。对T_1和T_2纤维素含量测定结果发现,转基因植株和受体纤维素含量无差异;T_1和T_2代株系棉纤维样品进行HVI900(ICC标准)品质检测,对纯合株系的分析表明:转基因棉花纤维长度和比强度较对照相比有明显降低,而伸长率显著增加。由此推测,GhBG基因的过量表达对纤维长度和比强度有影响。
Thousands of genes play role during fiber development,though the identification and function research of these genes was initiated recently,the progress was very quick.In this paper,we selected 10 preferentially expressed genes for fiber development which had reported previously and 14 cotton varieties(or strains) with significantly different fiber quality.Using Q-PCR technique to evaluate the relative expression value of 10 tested genes in different fiber developmental stages in 14 cotton varieties(or strains),in order to study the relationship between temporal and spatial expression of these genes and fiber quality traits.The analysis of expression profile of these genes in different cotton varieties(or strains) indicate:GhExp1,GhPL and GhCIPK1 gene preferentially express during fiber elongation;GhRacA and GhRacB gene have high expression level in earlier stage of fiber elongation and the thickening period of secondary cell wall;The predominant expression periods of GhACT1 are fiber elongation and the secondary cell wall thickening period;Two cellulose synthase genes(GhCelA1 and GhCelA3) preferentially express during late stage of fiber elongation and the thickening period of secondary cell wall;GhSus1 have high expression level during thickening period of secondary cell wall and fiber elongation stage, and GhSusA1 gene preferentially express during fiber initiation stage and the earlier stage of fiber elongation.For most genes,the expression value in low expression level period has significant correlation with fiber quality,while no significant correlation is detected when the expression of these genes at the peak except GhRacA gene.The expression level of GhExp1 in 20DPA fiber has a significant negative correlation with fiber strength and uniformity and it also has a significant positive correlation with fiber elongation;the expression level of GhPL gene in 23DPA has a significant negative correlation with fiber length;the expression level of GhRacA gene in 5DPA and 23DPA both have a high significant positive correlation with elongation;the expression level of GhRacB gene in 10DPA has a significant negative correlation with length;the expression level of GhCelA1 gene in 5DPA has a significant positive correlation with length and a significant negative correlation with Mic value;the expression level of GhCelA1 gene in 10DPA has a significant negative correlation with fiber length and a significant positive correlation with micronaire value and a high significant correlation with elongation;the expression level of GhCIPK1,GhACT1,GhSus1,GhSusA1and GhCelA3 gene have no significant correlation with fiber quality traits.
To characterize the possible function ofβ-1,4-glucosidase(GhBG) in cotton fiber,in this study,the sense coding sequence of the GhBG gene was fused with fiber-specific E6 promoter to build the constructs of sense gene,and with which to transform into Simian3 via Agrobacterium-mediated transformation.PCR amplification of the regenerated plants with primers of marker gene and target gene indicated that 56 plants from 6 clones were positive,of which 48 were survived by grafting.Southern blot analysis of the transgenic cotton plants showed that the introduction of GhBG gene into cotton genome was successful.Genetic Analysis of 17 T_1 and T_2 regenerated plants from 6 different transgenic lines by PCR analysis and Kanamycin-resistance assay indicated that the segregation ratio of GhBG gene in 6 transgenic lines were inherited in a classical Mendelian manner(3:1) and one pure line(504-5) was screened.For analyzing the expression level of this pure line, 0DPA,5DPA,10DPA,15DPA,20DPA and 23DPA ovule or fiber RNA of pure line was extracted,with Simian3 as control.The analysis of Q-PCR results showed that the expression level of GhBG gene in 5DPA,10DPA and 20DPA ovule or fiber of pure line was higher than that of control and the differences were significant.The result of mature celullose content test of T_1 and T_2 indicted that there was no significant difference between transgenic lines and control.Analysis of fiber quality traits showed that the fiber length and strength in pure line decreased and fiber elongation percentage increased.These results demonstrate that the overexpression of GhBG gene have effect to fiber length and strength.
引文
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