陆地棉转录因子的染色体定位
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摘要
棉花(Gossypium spp.)是世界上最重要的天然纤维作物。集纤维、蛋白、油用于一体。棉属50个种中有4个栽培种可生产纺织用纤维,分别是亚洲棉(G. arboreum)、草棉(G. herbaceum)、陆地棉(G. hirsutum)和海岛棉(G. barbadense)。其中陆地棉(G. hirsutum)占世界棉花总产量的95%以上。
我国不仅是一个产棉大国,也是用棉大国,棉花生产在国民经济中具有极其重要的地位。棉纤维具有吸湿、通气、保湿、柔软和不带静电等优点,因此越来越受到人们的喜爱,棉花的需求量也在日益增长。但低温、干旱、盐碱以及病虫害等生物和非生物胁迫因子都是限制棉花生长发育和纤维产量的重要因素。转录因子在植物生长发育、防卫反应和逆境胁迫应答过程中扮演着非常重要的角色。
转录因子(transcription factor, TF)又称反式作用因子(trans-acting factor),它与真核生物基因启动子区域中的顺式作用元件(cis-acting factor)发生特异性结合,并与其它相关蛋白互作,调节基因的表达强度或控制基因的时空特异性表达,应答激素刺激和外界环境胁迫。在作物分子育种中,与导入或改良个别功能基因的方法相比,导入或改良一个转录因子是提高作物产量、品质、抗逆性更为有效的方法和途径。转录因子的染色体定位对了解转录因子基因的染色体分布特点,准确把握基因的结构、功能和进化关系具有重要意义。
本研究利用北京大学生物信息中心鉴定的陆地棉转录因子DNA序列和NCBI(National Center for Biotechnology Information)数据库棉花MYB转录因子DNA序列设计SSR引物,并进行转录因子的染色体定位。这一方面可以增加棉花遗传作图标记,另一方面为进一步解析转录因子在棉花产量、纤维品质以及逆境抗性中的作用奠定基础。本研究结果如下：
1引物多态性分析
利用1116条陆地棉转录因子和65个棉花转录因子DNA序列设计的1592对SSR引物,筛选亲本渝棉1号与中棉所35(CRl35)、T586和7235,共获得74对多态性引物,占总引物的4.6%。其中,渝棉1号与中棉所35间25对多态性引物,占总引物的1.6%；渝棉1号与T586间33对多态性引物,占总引物的2.1%；渝棉1号与7235间36对多态性引物,占总引物的2.3%。74对多态性引物涵盖26个转录因子家族的69个转录因子。
2多态性引物的群体标记基因型检测
以74对多态性引物分别检测3个重组近交系群体,共获得101个位点。其中,25对引物检测(渝棉1号×中棉所35)群体,获得25个位点；36对引物检测(渝棉1号x7235)群体,获得41个位点；33对引物检测(渝棉1号xT586)群体,获得35个位点。
3转录因子基因染色体定位
对所获得的101个位点与本实验室前期对(渝棉1号×中棉所35)、(渝棉1号x7235)和(渝棉1号xT586)重组近交系群体定位的SSR标记位点进行遗传连锁分析,将其中92个位点定位于棉花23条染色体,9个位点未定位。(渝棉1号×中棉所35)群体定位23个位点,2个位点未定位；(渝棉1号×7235)群体定位39个位点,2个位点未定位；(渝棉1号×T586)群体定位30个位点,5个位点未定位。
4转录因子的家族与基因组分布
本研究涉及26个转录因子家族的69个成员,最少涉及某一转录因子家族的某1个转录因子(AP2、ARR-B等),最多的MYB转录因子家族达到9个转录因子。定了92个位点,其中32个属于A染色体亚组,60个属于D染色体亚组。
Cotton (Gossypium spp.) is the world's most important natural fiber crops. The genus Gossypium comprises approximately 45 diploid and 5 tetraploid species, including 4 cultivated species, G. arboreum, G. herbaceum, G. hirsutum and G. barbadense, which produce textile fiber. Of the cultivated species, G. hirsutum supplies over 95% of the world's total fiber production.
China is not only a major cotton producing country, but also a major cotton consuming country. Cotton has an extremely important position in the national economy. The unique feature of absorbing moisture, aeration, soft and static electricity exception are loved by people more and more, and the demand for cotton is growing day by day. However, cotton growth and yield are limited by biotic or abiotic stress, such as hypothermia, drought, salinity, pests and diseases. Transcription factors play a very important role in pant growth, stress response process and adversity defensing procedure.
Transcription factor (TF) is also called trans-acting factor which can specificly bind with the cis-acting factor in the promoter region, interact with other protein that regulate gene expression intensity and even control the spatial and temporal specificity of gene expression, and response to hormone stimulation and the external environment stress. To improve crop yield, quanlity and resistance to stress in molecular breeding, compared to transfering or modifying an individual functional gene, transfering or modifying a transcription factor is more effective. Manipulation of a transcription factor can prompt numbers of functional genes to play a role, so as to achieve access to comprehensive improvement of plant traits. Chromosomal localization of transcription factor has a great significance to understand the distribution characteristics of transcription factor, the accurate gene structure, function and evolution.
The present study designed SSR primer according to Gossypium hirsutum transcription factor DNA sequences in Plant Transcription Factor Databases (PTFD) and Gossypium MYB transcription factor DNA sequences in National Center for Biotechnology Information (NCBI), and mapped the polymorphic SSR in upland cotton. The results can increase the cotton genetic mapping markers, and lay the foundation for further resolving the role of transcription factors in the cotton yield, fiber quality and stress tolerance. The results are as following:
1 Primer polymorphism
A total of 1592 SSR primer pairs designed from 1116 Gossypium hirsutum transcription factor DNA sequences in PTFD and 65 transcription factor DNA sequences in NCBI were used to screen the polymorphic primers between upland cotton cultivars/lines Yumian 1,7235, CCRI35 and T586. A total of 74 pairs of polymorphic primers were obtained, accounting for 4.6% of the total primer pairs. The polymorphic primers included 25 polymorphic primers between Yumian 1 and CRI 35,33 between Yumian 1 and T586, 36 between Yumian 1 and 7235, accounting for 1.6%,2.1% and 2.3% of the total primers, respectively. Seventy-four pairs of polymorphic primers cover 69 transcription factors of the 26 transcription factor families.
2 Genotyping RIL populations
Three recombinant inbred line populations were genotyped with the primer pairs showing polymorphism between two parents and 101 polymorphic loci were detected. Among them, 25 primer pairs obtained 25 loci in population (Yumian1×CRI35),36 primer pairs obtained 41 loci in population (Yumian1×7235), and 33 primer pairs obtained 35 loci in population (Yumianl×T586).
3 Localizing transcription factors
The 101 transcription factor SSR loci, together with other SSR loci mapped on upland cotton linkage map in our laboratory, were used to conduct on genetic linkage analysis, and 92 loci were mapped on 23 chromosomes. Twenty-three of 25 loci were mapped on chromosome in population (Yumian 1×CRI35) population,30 of 35 loci were mapped on chromosome in population (Yumian 1×T586), and 39 of 41 loci were mapped in population (Yumian 1×7235). 4 Distribution of transcription factor genes
The mapped 26 transcription factor families included 69 members, and involved one to 19 members from each family. A total of 92 loci were mapped on 23 chromosomes, including 32 loci cotton A-genome and 60 on D-genome from tetraploid cotton.
我国不仅是一个产棉大国,也是用棉大国,棉花生产在国民经济中具有极其重要的地位。棉纤维具有吸湿、通气、保湿、柔软和不带静电等优点,因此越来越受到人们的喜爱,棉花的需求量也在日益增长。但低温、干旱、盐碱以及病虫害等生物和非生物胁迫因子都是限制棉花生长发育和纤维产量的重要因素。转录因子在植物生长发育、防卫反应和逆境胁迫应答过程中扮演着非常重要的角色。
转录因子(transcription factor, TF)又称反式作用因子(trans-acting factor),它与真核生物基因启动子区域中的顺式作用元件(cis-acting factor)发生特异性结合,并与其它相关蛋白互作,调节基因的表达强度或控制基因的时空特异性表达,应答激素刺激和外界环境胁迫。在作物分子育种中,与导入或改良个别功能基因的方法相比,导入或改良一个转录因子是提高作物产量、品质、抗逆性更为有效的方法和途径。转录因子的染色体定位对了解转录因子基因的染色体分布特点,准确把握基因的结构、功能和进化关系具有重要意义。
本研究利用北京大学生物信息中心鉴定的陆地棉转录因子DNA序列和NCBI(National Center for Biotechnology Information)数据库棉花MYB转录因子DNA序列设计SSR引物,并进行转录因子的染色体定位。这一方面可以增加棉花遗传作图标记,另一方面为进一步解析转录因子在棉花产量、纤维品质以及逆境抗性中的作用奠定基础。本研究结果如下：
1引物多态性分析
利用1116条陆地棉转录因子和65个棉花转录因子DNA序列设计的1592对SSR引物,筛选亲本渝棉1号与中棉所35(CRl35)、T586和7235,共获得74对多态性引物,占总引物的4.6%。其中,渝棉1号与中棉所35间25对多态性引物,占总引物的1.6%；渝棉1号与T586间33对多态性引物,占总引物的2.1%；渝棉1号与7235间36对多态性引物,占总引物的2.3%。74对多态性引物涵盖26个转录因子家族的69个转录因子。
2多态性引物的群体标记基因型检测
以74对多态性引物分别检测3个重组近交系群体,共获得101个位点。其中,25对引物检测(渝棉1号×中棉所35)群体,获得25个位点；36对引物检测(渝棉1号x7235)群体,获得41个位点；33对引物检测(渝棉1号xT586)群体,获得35个位点。
3转录因子基因染色体定位
对所获得的101个位点与本实验室前期对(渝棉1号×中棉所35)、(渝棉1号x7235)和(渝棉1号xT586)重组近交系群体定位的SSR标记位点进行遗传连锁分析,将其中92个位点定位于棉花23条染色体,9个位点未定位。(渝棉1号×中棉所35)群体定位23个位点,2个位点未定位；(渝棉1号×7235)群体定位39个位点,2个位点未定位；(渝棉1号×T586)群体定位30个位点,5个位点未定位。
4转录因子的家族与基因组分布
本研究涉及26个转录因子家族的69个成员,最少涉及某一转录因子家族的某1个转录因子(AP2、ARR-B等),最多的MYB转录因子家族达到9个转录因子。定了92个位点,其中32个属于A染色体亚组,60个属于D染色体亚组。
Cotton (Gossypium spp.) is the world's most important natural fiber crops. The genus Gossypium comprises approximately 45 diploid and 5 tetraploid species, including 4 cultivated species, G. arboreum, G. herbaceum, G. hirsutum and G. barbadense, which produce textile fiber. Of the cultivated species, G. hirsutum supplies over 95% of the world's total fiber production.
China is not only a major cotton producing country, but also a major cotton consuming country. Cotton has an extremely important position in the national economy. The unique feature of absorbing moisture, aeration, soft and static electricity exception are loved by people more and more, and the demand for cotton is growing day by day. However, cotton growth and yield are limited by biotic or abiotic stress, such as hypothermia, drought, salinity, pests and diseases. Transcription factors play a very important role in pant growth, stress response process and adversity defensing procedure.
Transcription factor (TF) is also called trans-acting factor which can specificly bind with the cis-acting factor in the promoter region, interact with other protein that regulate gene expression intensity and even control the spatial and temporal specificity of gene expression, and response to hormone stimulation and the external environment stress. To improve crop yield, quanlity and resistance to stress in molecular breeding, compared to transfering or modifying an individual functional gene, transfering or modifying a transcription factor is more effective. Manipulation of a transcription factor can prompt numbers of functional genes to play a role, so as to achieve access to comprehensive improvement of plant traits. Chromosomal localization of transcription factor has a great significance to understand the distribution characteristics of transcription factor, the accurate gene structure, function and evolution.
The present study designed SSR primer according to Gossypium hirsutum transcription factor DNA sequences in Plant Transcription Factor Databases (PTFD) and Gossypium MYB transcription factor DNA sequences in National Center for Biotechnology Information (NCBI), and mapped the polymorphic SSR in upland cotton. The results can increase the cotton genetic mapping markers, and lay the foundation for further resolving the role of transcription factors in the cotton yield, fiber quality and stress tolerance. The results are as following:
1 Primer polymorphism
A total of 1592 SSR primer pairs designed from 1116 Gossypium hirsutum transcription factor DNA sequences in PTFD and 65 transcription factor DNA sequences in NCBI were used to screen the polymorphic primers between upland cotton cultivars/lines Yumian 1,7235, CCRI35 and T586. A total of 74 pairs of polymorphic primers were obtained, accounting for 4.6% of the total primer pairs. The polymorphic primers included 25 polymorphic primers between Yumian 1 and CRI 35,33 between Yumian 1 and T586, 36 between Yumian 1 and 7235, accounting for 1.6%,2.1% and 2.3% of the total primers, respectively. Seventy-four pairs of polymorphic primers cover 69 transcription factors of the 26 transcription factor families.
2 Genotyping RIL populations
Three recombinant inbred line populations were genotyped with the primer pairs showing polymorphism between two parents and 101 polymorphic loci were detected. Among them, 25 primer pairs obtained 25 loci in population (Yumian1×CRI35),36 primer pairs obtained 41 loci in population (Yumian1×7235), and 33 primer pairs obtained 35 loci in population (Yumianl×T586).
3 Localizing transcription factors
The 101 transcription factor SSR loci, together with other SSR loci mapped on upland cotton linkage map in our laboratory, were used to conduct on genetic linkage analysis, and 92 loci were mapped on 23 chromosomes. Twenty-three of 25 loci were mapped on chromosome in population (Yumian 1×CRI35) population,30 of 35 loci were mapped on chromosome in population (Yumian 1×T586), and 39 of 41 loci were mapped in population (Yumian 1×7235). 4 Distribution of transcription factor genes
The mapped 26 transcription factor families included 69 members, and involved one to 19 members from each family. A total of 92 loci were mapped on 23 chromosomes, including 32 loci cotton A-genome and 60 on D-genome from tetraploid cotton.
引文
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