长武134小麦GAPDH启动子克隆及序列分析
摘要
本文以抗旱性强的小麦长武134为试验材料,通过对长武134小麦进行-1.2MPaPEG胁迫处理,利用基因组DNA纯化试剂盒提取胁迫处理48h的小麦黄化芽基因组DNA。以提取的基因组DNA为模板,利用Genome Walking的方法克隆小麦甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)上游序列,长度为1071bp,并对其功能进行生物信息学分析,为GAPDH的研究提供基础。同时,以长武134、晋麦47、西农2208和陕253为材料对其成熟胚的组织培养进行了研究,为GAPDH构建植物高效表达载体后转基因建立高效的小麦成熟胚植株再生体系。
结果显示:
1.利用Takara基因组DNA纯化试剂盒提取小麦基因组DNA符合克隆GAPDH启动子试验要求,以提取的小麦基因组DNA为模板克隆GAPDH启动子,测序后得到翻译起始密码子上游1071bp序列。在Genebank中对该序列BLAST分析,未发现同源性高的序列,推测该片段为一新序列。
2.对获得的新序列进行启动子预测和顺式作用元件分析,发现该序列中可能包含4个启动子区域;在翻译起始密码子ATG上游存在启动子基本结构元件TATA-box1个和CAAT框7个及多种顺式元件,其中WRKY、MYB、MYC、GT-1、HSE和ABRE元件能够应答ABA、干旱等非生物胁迫逆境因子。
3.小麦成熟胚愈伤组织在诱导培养基中2,4-D浓度为2-4 mg/L时的出愈率高,愈伤组织的品质好,但之间差异并非极显著,出愈率都在84.5%以上。而且胚愈伤组织的出愈率与愈伤组织的品质及愈伤组织生长状况三者之间没有直接关系。
The Changwu134 wheat with drought stress were used in this paper. Genomic DNA was isolated from Changwu134 wheat seeding was treated with -1.2MPa PEG6000 solution for 48h by DNAiso Reagent Kit. Through the Genome Walking method and TA cloning, the GAPDH ( glyceraldehyde-3-phosphate dehydrogenase ) upstream fragment was obtained in the wheat seeding, and the length of the sequence is 1071bp. Meanwhile, explant of mature embryos of 4 different wheat genetypes were cultured in differention media to study the induction of the callus and genetic difference of plants regeneration from different genetypes.
1. The genomic DNA was extracted meets the GAPDH promoter cloning test requirements. The length of the GAPDH promoter fragment was obtained is 1071bp. Sequence analysis indicated that this fragment had no alignment with other promoters. So we speculated that that this fragment was a new fragment.
2. Analysis of target fragment in promoter prediction showed that it contained four promoter motifs and one TATA-box, seven CAAT-box, and several cis-action elements such as WRKY、MYB、MYC、GT-1、HSE and ABRE can responsive to ABA, drought, hight salt and pathology.
3. The results of wheat mature embryos callus induced and differentiation indicate that 2,4-D (2 mg/L-4 mg/L) was more advantageous to the induction of callus differentiation. And callus inducement rate, quality and fresh weight haven’t direct relatonship with each other.
结果显示:
1.利用Takara基因组DNA纯化试剂盒提取小麦基因组DNA符合克隆GAPDH启动子试验要求,以提取的小麦基因组DNA为模板克隆GAPDH启动子,测序后得到翻译起始密码子上游1071bp序列。在Genebank中对该序列BLAST分析,未发现同源性高的序列,推测该片段为一新序列。
2.对获得的新序列进行启动子预测和顺式作用元件分析,发现该序列中可能包含4个启动子区域;在翻译起始密码子ATG上游存在启动子基本结构元件TATA-box1个和CAAT框7个及多种顺式元件,其中WRKY、MYB、MYC、GT-1、HSE和ABRE元件能够应答ABA、干旱等非生物胁迫逆境因子。
3.小麦成熟胚愈伤组织在诱导培养基中2,4-D浓度为2-4 mg/L时的出愈率高,愈伤组织的品质好,但之间差异并非极显著,出愈率都在84.5%以上。而且胚愈伤组织的出愈率与愈伤组织的品质及愈伤组织生长状况三者之间没有直接关系。
The Changwu134 wheat with drought stress were used in this paper. Genomic DNA was isolated from Changwu134 wheat seeding was treated with -1.2MPa PEG6000 solution for 48h by DNAiso Reagent Kit. Through the Genome Walking method and TA cloning, the GAPDH ( glyceraldehyde-3-phosphate dehydrogenase ) upstream fragment was obtained in the wheat seeding, and the length of the sequence is 1071bp. Meanwhile, explant of mature embryos of 4 different wheat genetypes were cultured in differention media to study the induction of the callus and genetic difference of plants regeneration from different genetypes.
1. The genomic DNA was extracted meets the GAPDH promoter cloning test requirements. The length of the GAPDH promoter fragment was obtained is 1071bp. Sequence analysis indicated that this fragment had no alignment with other promoters. So we speculated that that this fragment was a new fragment.
2. Analysis of target fragment in promoter prediction showed that it contained four promoter motifs and one TATA-box, seven CAAT-box, and several cis-action elements such as WRKY、MYB、MYC、GT-1、HSE and ABRE can responsive to ABA, drought, hight salt and pathology.
3. The results of wheat mature embryos callus induced and differentiation indicate that 2,4-D (2 mg/L-4 mg/L) was more advantageous to the induction of callus differentiation. And callus inducement rate, quality and fresh weight haven’t direct relatonship with each other.
引文
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