基于芯片杂交结果克隆疏叶骆驼刺、苦马豆相关基因的研究
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摘要
疏叶骆驼刺(Alhagi sparsifolia Shap.)是豆科骆驼刺属(Alhagi Gagneb.)多年生草本植物,耐旱、耐盐碱和抗涝强,是干旱区的优良牧草,也是很好的蜜源植物和药用植物。苦马豆[Swainsona salsula Taub. (Sphaerophy salsula (Pall.) DC.)]又名羊尿泡、马尿泡,是多年生豆科苦马豆属植物,是耐盐碱、抗涝强的植物,具有改良土壤及抗癌调节免疫活性的作用。
本实验通过对疏叶骆驼刺、苦马豆与截形苜蓿芯片杂交结果的分析,结合截形苜蓿生物信息学数据,从疏叶骆驼刺中筛选出非共生血红蛋白nSHb和甲酸脱氢酶FDH两个基因,从苦马豆中筛选出异黄酮还原酶IFR基因。
我们分别提取疏叶骆驼刺和苦马豆的总RNA,利用oligo dT反转录为cDNA,然后根据截型苜蓿GenBank中nSHb、FDH和IFR的cDNA序列设计引物,采用RT-PCR技术扩增出三个基因的全长cDNA序列,分别命名为AsnSHb,AsFDH和StIFR。其中,AsnSHb基因长度为483bp,编码160个氨基酸;AsFDH长度为1170bp,编码389个氨基酸;StIFR长度为957bp,编码318个氨基酸,通过与苜蓿序列的同源性比较,发现基因序列的同源性分别为92.50%、89.41%和81.82%,翻译蛋白的同源性分别为91.51%、92.03%和83.39%。并且构建了表达载体,通过农杆菌转化的方法将这三个基因分别转到烟草和拟南芥中,为进一步验证nSHb、FDH和IFR基因所编码蛋白的相关功能奠定基础。
设计5’端带有BglⅡ和3’端带有SpeⅠ酶切位点的引物,利用PCR技术分别扩增AsnSHb、AsFDH和StIFR基因编码区,然后插入到pCAMBIA1_30_2载体的BglⅡ和SpeⅠ位点上构建表达载体,双酶切鉴定后将该载体转入农杆菌EHA105,用利福平抗生素进行筛选,并且通过农杆菌转化的方法分别将这三个基因转到烟草和拟南芥中。
Alhagi sparsifolia Shap. classified to Alhagi Gagneb. is perennial plant as bee and medical plant and it is tolerant to drought, salt and waterlogging as forage. While Swainsona salsula Taub. classified to Swainsonia Salisb form Leguminosae. Has the anticancer function and immunoregulatory activity. As a neutral plant tolerated to salt, it also can improve soil conditions.
Through analysis Alhagi sparsifolia Shap. and Swainsona salsula Taub. hybridizing the chip hybridization results of Medicago truncatula by Bioinformatic, we screened two genes non-symbiotic hemoglobin (nSHb) and formate dehydrogenase (FDH) from Alhagi sparsifolia Shap.and one gene isoflavone reductase (IFR) from SwainsonasalsulaTaub.
We extracted total RNA from Alhagi sparsifolia and Swainson asalsula Taub, and reverse transcribed it to cDNA using oligo dT, then designed the primers according to the nSHb, FDH and IFR gene sequence in Medicago truncatula genebank, amplified the three full length cDNAs using RT-PCR technique, named them AsnSHb, AsFDH and StIFR.. As follows :The length of AsnSHb is 483bp encoding 160 amino acids, AsFDH is 1170 bp encoding 389 amino acids and StIFR is 957bp encoding 318 amino acids. Through blasting with Medicago sativa database the homology of nucleotide is 92.50%, 89.41% and 81.82% respectively, the homology of amino acids is 91.51%, 92.03% and 83.39% respectively. Furthermore, we constructed three expression vectors and transformed them to Nicotiana tabacum and Arabidopsis thaliana through Agrobacterium transformation. Our results would lay the foundation of molecular information to study the function of the protein coded by nSHb, FDH and IFR.
We devised the primers with BglⅡand SpeⅠrestriction enzyme sites, amplified ORF of three cDNAs using PCR, then inserted them into the vector pCAMBIA1_30_2 by BglⅡand SpeⅠsites. After identifying the three recombinant plasmids using BglⅡand SpeⅠrestriction enzyme digestion we transformed the plasmids into Agrobacterium rhizogenes EHA105 and screened by rifampicin. Furthermore, we transformed these three genes to Nicotiana tabacum and Arabidopsis thaliana by the way of Agrobacterium transformation.
本实验通过对疏叶骆驼刺、苦马豆与截形苜蓿芯片杂交结果的分析,结合截形苜蓿生物信息学数据,从疏叶骆驼刺中筛选出非共生血红蛋白nSHb和甲酸脱氢酶FDH两个基因,从苦马豆中筛选出异黄酮还原酶IFR基因。
我们分别提取疏叶骆驼刺和苦马豆的总RNA,利用oligo dT反转录为cDNA,然后根据截型苜蓿GenBank中nSHb、FDH和IFR的cDNA序列设计引物,采用RT-PCR技术扩增出三个基因的全长cDNA序列,分别命名为AsnSHb,AsFDH和StIFR。其中,AsnSHb基因长度为483bp,编码160个氨基酸;AsFDH长度为1170bp,编码389个氨基酸;StIFR长度为957bp,编码318个氨基酸,通过与苜蓿序列的同源性比较,发现基因序列的同源性分别为92.50%、89.41%和81.82%,翻译蛋白的同源性分别为91.51%、92.03%和83.39%。并且构建了表达载体,通过农杆菌转化的方法将这三个基因分别转到烟草和拟南芥中,为进一步验证nSHb、FDH和IFR基因所编码蛋白的相关功能奠定基础。
设计5’端带有BglⅡ和3’端带有SpeⅠ酶切位点的引物,利用PCR技术分别扩增AsnSHb、AsFDH和StIFR基因编码区,然后插入到pCAMBIA1_30_2载体的BglⅡ和SpeⅠ位点上构建表达载体,双酶切鉴定后将该载体转入农杆菌EHA105,用利福平抗生素进行筛选,并且通过农杆菌转化的方法分别将这三个基因转到烟草和拟南芥中。
Alhagi sparsifolia Shap. classified to Alhagi Gagneb. is perennial plant as bee and medical plant and it is tolerant to drought, salt and waterlogging as forage. While Swainsona salsula Taub. classified to Swainsonia Salisb form Leguminosae. Has the anticancer function and immunoregulatory activity. As a neutral plant tolerated to salt, it also can improve soil conditions.
Through analysis Alhagi sparsifolia Shap. and Swainsona salsula Taub. hybridizing the chip hybridization results of Medicago truncatula by Bioinformatic, we screened two genes non-symbiotic hemoglobin (nSHb) and formate dehydrogenase (FDH) from Alhagi sparsifolia Shap.and one gene isoflavone reductase (IFR) from SwainsonasalsulaTaub.
We extracted total RNA from Alhagi sparsifolia and Swainson asalsula Taub, and reverse transcribed it to cDNA using oligo dT, then designed the primers according to the nSHb, FDH and IFR gene sequence in Medicago truncatula genebank, amplified the three full length cDNAs using RT-PCR technique, named them AsnSHb, AsFDH and StIFR.. As follows :The length of AsnSHb is 483bp encoding 160 amino acids, AsFDH is 1170 bp encoding 389 amino acids and StIFR is 957bp encoding 318 amino acids. Through blasting with Medicago sativa database the homology of nucleotide is 92.50%, 89.41% and 81.82% respectively, the homology of amino acids is 91.51%, 92.03% and 83.39% respectively. Furthermore, we constructed three expression vectors and transformed them to Nicotiana tabacum and Arabidopsis thaliana through Agrobacterium transformation. Our results would lay the foundation of molecular information to study the function of the protein coded by nSHb, FDH and IFR.
We devised the primers with BglⅡand SpeⅠrestriction enzyme sites, amplified ORF of three cDNAs using PCR, then inserted them into the vector pCAMBIA1_30_2 by BglⅡand SpeⅠsites. After identifying the three recombinant plasmids using BglⅡand SpeⅠrestriction enzyme digestion we transformed the plasmids into Agrobacterium rhizogenes EHA105 and screened by rifampicin. Furthermore, we transformed these three genes to Nicotiana tabacum and Arabidopsis thaliana by the way of Agrobacterium transformation.
引文
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