烟草吸钾相关基因克隆与表达调控研究
摘要
烟草钾素营养不但在烟叶生长、发育过程中影响烟叶的抗病性、抗逆性和适烤性,直接影响烟叶的香气质、香气量和燃烧性,还与烟气中焦油、苯并芘等有害物质含量有着密切的相关性,烟叶含钾量是烟叶重要的品质指标。调查表明我国烟叶含钾量显著低于美国、津巴布韦和巴西等国际优质烟叶,多年来,学者们曾尝试从增加钾肥施用量、改善施肥方法等方面提高烟叶含钾量,但收效甚微,烟叶含钾量过低一直是困扰我国烟区的重大技术难题。
本研究克隆了拟南芥钾离子转运蛋白基因AtKup1、Na~+/H~+反向转运体基因AtNHX1和无机焦磷酸酶基因AVP2,分别构建高效植物表达载体,通过根癌农杆菌介导法转化烟草,分析了3类基因转化烟草幼根中5种内源吸钾相关基因(烟草钾通道、K~+转运蛋白、无机焦磷酸酶、细胞膜H~+泵和液泡膜H~+泵)转录水平,结果表明:在转AtKup1基因烟草幼根中,烟草K~+转运蛋白基因NtHAK1转录显著降低,细胞膜H~+-ATPase基因显著增加,其它基因转录没有显著变化;在过量表达AtNHX1基因的烟草幼根中,烟草钾离子转运蛋白基因NtHAK1的转录水平显著降低,H~+-ATPase基因NHA1和VAG1显著增加;而焦磷酸酶基因NVP1的转录稍有增加,钾通道基因NKT1转录变化不大;过量表达AVP2基因使烟草幼根中的烟草液泡膜H~+-ATPase基因VAG1和液泡膜H~+-PPase基因NVP1的转录水平降低,K~+转运体基因NtHAK1基因显著增加,而钾通道基因NKT1稍有增加,细胞质膜H~+-ATPase基因NHA1无显著变化。烟叶内在化学成分分析结果表明:三种基因的过量表达均可不同程度地增强烟株根系的吸钾能力,提高烟叶含钾量。
为增强外源吸钾基因的表达活性,本研究采用DNA改组方法进行AtKup1基因的体外分子进化,建立AtKup1基因突变库,筛选吸钾活性增强的AtKup1基因的突变型,并导入烟草进行表达分析,获得吸钾能力显著提高的转化材料。通过对拟南芥等不同物种间的K~+转运蛋白基因KT/KUP/HAK、钾通道基因、质子泵(H~+-ATPase)基因和H~+转运无机焦磷酸酶基因的同源性比对分析,采用兼并引物PCR扩增和RACE方法分别获得了烟草4个吸钾相关基因的cDNA全序列(NrHAK1, NKC1, NVP1和NtHAK1),并将所获得的NrHAK1基因导入酿酒酵母钾吸收缺陷型菌株进行功能性研究,结果发现:NrHAK1基因可以使酵母钾吸收缺陷型菌株恢复吸钾能力。烟草不同组织的转录水平分析发现:NrHAK1基因在烟苗幼根转录量最大,证实该基因与烟草根系吸钾有关。
应用荧光定量PCR原理,通过优化引物设计、qRT-PCR反应体系和反应条件,选择适宜的看家基因,建立了适于植物分子营养研究的mRNA定量方法,并利用这一方法研究了K~+、Ca2~+无机营养缺乏和Na~+、NH4~+高盐胁迫条件下,烟草K~+通道基因NKT1、K~+转运蛋白基因NtHAK1、无机焦磷酸酶基因NVP1、细胞质膜质子泵基因NHA1和液泡膜质子泵基因VAG1的转录水平。结果表明:K~+饥饿条件下,K~+转运蛋白基因NtHAK1基因转录量增加, V-PPase基因NVP1的转录降低;在缺钙条件下,钾通道基因NKT1、烟草K~+转运蛋白基NtHAK1,编码P-ATPase的G亚基VAG1基因和V-ATPase的NHA1基因转录显著下降;在高浓度Na~+胁迫条件下,植物根系钾离子转运蛋白基因NtHAK1、质膜质子泵P-H~+-ATPase基因NHA1和液泡膜质子泵V-H~+-ATPase基因VAG1转录显著增加,钾通道基因NKT1降低。试验还发现环境中高浓度的NH4~+使钾离子转运蛋白基因显著降低,V-ATPase基因NHA1和V-PPase基因NVP1均有所增加。
Except for affecting the disease resistance and stress responses in tobacco growing periods, the potassium nutrition has close relations with quantity and quality of flavor, burn capacity and the amount of harmful materials such as tar and benzopyrene. The potassium content in tobacco leavies is an important index for high quality tobacco leaf. Surveys have confirmed that potassium content of domestic tobacco leaf is much less than that of international high quality tobacco leavies from USA, Zimbabwe and Brazil. Researchers from home and abroad have done many studies to increase the potassium contents in tobacco leaf through raising fertilizer amounts or improving fertilization methods in recent years. But unfortunately, all the works have failed to increase the potassium of tobacco leavies. Low potassium content in tobacco leaf is an important technique problem which puzzled growers in domestic tobacco growing areas.
Three genes including AtKup1 encoding the K~+ transporter, AtNHX1 encoding the Na~+/H~+ anti-porter and inorganic pyrophosphatase AVP2 have been cloned from Arabidopsis. The plant binary expression vectors of the 3 genes have been constructed and transformed into tobacco by the meditation of Agrobacterium tumefaciens. The transcript of 5 tobacco internal genes encoding K~+ channel, K~+ transporter, vacuole H~+-PPase, plasma H~+-ATPase and vacuole H~+-ATPase have been analyzed in transgenic tobacco plants. The results demonstrated that the transcript of NtHAK1 gene was reduced and that of NHA1 was increased significantly in the roots of the AtKup1 trans-formants. The transcript of NtHAK1 gene was also down regulated in the roots of AtNHX1 trans-formants, but the transcripts of NHA1 and VAG1 encoding H~+-ATPase were up regulated significantly and that of NVP1 encoding vacuole H~+-PPase was increased slightly. The transcript of VAG1 encoding H~+-ATPase and NVP1 encoding for vacuole H~+-PPase are down regulated and the transcript of NtHAK1 are up regulated significantly and that of K~+ channel NKT1 are increased slightly in the tobacco root over expressed of AVP2 gene. The chemical contents analysis of tobacco leaf indicated that the potassium uptake capacity of trans-formant roots were increased differently when the tobacco was genetically modified by the three genes above respectively and the potassium contents in the tobacco leaf were increased differently.
PCR-based DNA shuffling was carried out for AtKup1 gene to recombinatorial molecular evolution in vivo. The AtKup1 mutations with high capacity of K~+ uptake were screened from recombination PCR fragments libraries and introduced into TRK1 and TRK2 defective yeast strain.
By the bioinformatics analysis of genes encoding to K~+ high affinity transporter KT/HAK/KUP, potassium channel, H~+-ATPase and vacuole H~+-PPase from Arabidopsis and other species in NCBI. Four cDNA complete sequences incuding NrHAK1, NKC1, NVP1 and NtHAK1 were obtained through the methods of degenerate primers PCR and RACE, The nucleotide sequence have been deposited in the NCBI data base. Function characterization of NrHAK1 which was carried out by introduced it into the K~+ uptake genes TRK1 and TRK2 defective yeast strain demonstrated that NrHAK1 can complement yeast mutants defective in K~+ uptake. It was found that the mRNA of NrHAK1 was the most abundant in roots of Nicotiana rustica which suggest that NrHAK1 plays an important role in potassium uptaking of plant roots.
The qRT-PCR technique which suit for mRNA measurements in the molecular nutrition studies was established by performing the optimized experiments of primer designation, PCR systems and condition as well as house keeping gene selection. Transcript of five genes related to the potassium uptake including K~+ channel, K~+ transporter, cytoplasm H~+-ATPase, H~+-PPase and vacuole H~+-ATPase under different nutrition and salts stress conditions were analyzed by using qRT-PCR methods. It was found that the transcript of NtHAK1 and NHA1 were significantly stimulated and that of NVP1 was reduced when the K~+ starvation exist in external solution. The results also confirmed that the transcript of K~+ channel gene NKT1, K~+ transporter gene NtHAK1, V-H~+-ATPase G unit gene VAG1 and P-H~+-ATPase gene NHA1 were down regulated when the tobacco was Ca2~+ starved. The transcript of K~+ transporter gene NtHAK1 was induced by excessive Na~+ salinity stress, but that was inhibited when tobacco plant exposed to the solution contained 5 mmol/L NH4~+.
本研究克隆了拟南芥钾离子转运蛋白基因AtKup1、Na~+/H~+反向转运体基因AtNHX1和无机焦磷酸酶基因AVP2,分别构建高效植物表达载体,通过根癌农杆菌介导法转化烟草,分析了3类基因转化烟草幼根中5种内源吸钾相关基因(烟草钾通道、K~+转运蛋白、无机焦磷酸酶、细胞膜H~+泵和液泡膜H~+泵)转录水平,结果表明:在转AtKup1基因烟草幼根中,烟草K~+转运蛋白基因NtHAK1转录显著降低,细胞膜H~+-ATPase基因显著增加,其它基因转录没有显著变化;在过量表达AtNHX1基因的烟草幼根中,烟草钾离子转运蛋白基因NtHAK1的转录水平显著降低,H~+-ATPase基因NHA1和VAG1显著增加;而焦磷酸酶基因NVP1的转录稍有增加,钾通道基因NKT1转录变化不大;过量表达AVP2基因使烟草幼根中的烟草液泡膜H~+-ATPase基因VAG1和液泡膜H~+-PPase基因NVP1的转录水平降低,K~+转运体基因NtHAK1基因显著增加,而钾通道基因NKT1稍有增加,细胞质膜H~+-ATPase基因NHA1无显著变化。烟叶内在化学成分分析结果表明:三种基因的过量表达均可不同程度地增强烟株根系的吸钾能力,提高烟叶含钾量。
为增强外源吸钾基因的表达活性,本研究采用DNA改组方法进行AtKup1基因的体外分子进化,建立AtKup1基因突变库,筛选吸钾活性增强的AtKup1基因的突变型,并导入烟草进行表达分析,获得吸钾能力显著提高的转化材料。通过对拟南芥等不同物种间的K~+转运蛋白基因KT/KUP/HAK、钾通道基因、质子泵(H~+-ATPase)基因和H~+转运无机焦磷酸酶基因的同源性比对分析,采用兼并引物PCR扩增和RACE方法分别获得了烟草4个吸钾相关基因的cDNA全序列(NrHAK1, NKC1, NVP1和NtHAK1),并将所获得的NrHAK1基因导入酿酒酵母钾吸收缺陷型菌株进行功能性研究,结果发现:NrHAK1基因可以使酵母钾吸收缺陷型菌株恢复吸钾能力。烟草不同组织的转录水平分析发现:NrHAK1基因在烟苗幼根转录量最大,证实该基因与烟草根系吸钾有关。
应用荧光定量PCR原理,通过优化引物设计、qRT-PCR反应体系和反应条件,选择适宜的看家基因,建立了适于植物分子营养研究的mRNA定量方法,并利用这一方法研究了K~+、Ca2~+无机营养缺乏和Na~+、NH4~+高盐胁迫条件下,烟草K~+通道基因NKT1、K~+转运蛋白基因NtHAK1、无机焦磷酸酶基因NVP1、细胞质膜质子泵基因NHA1和液泡膜质子泵基因VAG1的转录水平。结果表明:K~+饥饿条件下,K~+转运蛋白基因NtHAK1基因转录量增加, V-PPase基因NVP1的转录降低;在缺钙条件下,钾通道基因NKT1、烟草K~+转运蛋白基NtHAK1,编码P-ATPase的G亚基VAG1基因和V-ATPase的NHA1基因转录显著下降;在高浓度Na~+胁迫条件下,植物根系钾离子转运蛋白基因NtHAK1、质膜质子泵P-H~+-ATPase基因NHA1和液泡膜质子泵V-H~+-ATPase基因VAG1转录显著增加,钾通道基因NKT1降低。试验还发现环境中高浓度的NH4~+使钾离子转运蛋白基因显著降低,V-ATPase基因NHA1和V-PPase基因NVP1均有所增加。
Except for affecting the disease resistance and stress responses in tobacco growing periods, the potassium nutrition has close relations with quantity and quality of flavor, burn capacity and the amount of harmful materials such as tar and benzopyrene. The potassium content in tobacco leavies is an important index for high quality tobacco leaf. Surveys have confirmed that potassium content of domestic tobacco leaf is much less than that of international high quality tobacco leavies from USA, Zimbabwe and Brazil. Researchers from home and abroad have done many studies to increase the potassium contents in tobacco leaf through raising fertilizer amounts or improving fertilization methods in recent years. But unfortunately, all the works have failed to increase the potassium of tobacco leavies. Low potassium content in tobacco leaf is an important technique problem which puzzled growers in domestic tobacco growing areas.
Three genes including AtKup1 encoding the K~+ transporter, AtNHX1 encoding the Na~+/H~+ anti-porter and inorganic pyrophosphatase AVP2 have been cloned from Arabidopsis. The plant binary expression vectors of the 3 genes have been constructed and transformed into tobacco by the meditation of Agrobacterium tumefaciens. The transcript of 5 tobacco internal genes encoding K~+ channel, K~+ transporter, vacuole H~+-PPase, plasma H~+-ATPase and vacuole H~+-ATPase have been analyzed in transgenic tobacco plants. The results demonstrated that the transcript of NtHAK1 gene was reduced and that of NHA1 was increased significantly in the roots of the AtKup1 trans-formants. The transcript of NtHAK1 gene was also down regulated in the roots of AtNHX1 trans-formants, but the transcripts of NHA1 and VAG1 encoding H~+-ATPase were up regulated significantly and that of NVP1 encoding vacuole H~+-PPase was increased slightly. The transcript of VAG1 encoding H~+-ATPase and NVP1 encoding for vacuole H~+-PPase are down regulated and the transcript of NtHAK1 are up regulated significantly and that of K~+ channel NKT1 are increased slightly in the tobacco root over expressed of AVP2 gene. The chemical contents analysis of tobacco leaf indicated that the potassium uptake capacity of trans-formant roots were increased differently when the tobacco was genetically modified by the three genes above respectively and the potassium contents in the tobacco leaf were increased differently.
PCR-based DNA shuffling was carried out for AtKup1 gene to recombinatorial molecular evolution in vivo. The AtKup1 mutations with high capacity of K~+ uptake were screened from recombination PCR fragments libraries and introduced into TRK1 and TRK2 defective yeast strain.
By the bioinformatics analysis of genes encoding to K~+ high affinity transporter KT/HAK/KUP, potassium channel, H~+-ATPase and vacuole H~+-PPase from Arabidopsis and other species in NCBI. Four cDNA complete sequences incuding NrHAK1, NKC1, NVP1 and NtHAK1 were obtained through the methods of degenerate primers PCR and RACE, The nucleotide sequence have been deposited in the NCBI data base. Function characterization of NrHAK1 which was carried out by introduced it into the K~+ uptake genes TRK1 and TRK2 defective yeast strain demonstrated that NrHAK1 can complement yeast mutants defective in K~+ uptake. It was found that the mRNA of NrHAK1 was the most abundant in roots of Nicotiana rustica which suggest that NrHAK1 plays an important role in potassium uptaking of plant roots.
The qRT-PCR technique which suit for mRNA measurements in the molecular nutrition studies was established by performing the optimized experiments of primer designation, PCR systems and condition as well as house keeping gene selection. Transcript of five genes related to the potassium uptake including K~+ channel, K~+ transporter, cytoplasm H~+-ATPase, H~+-PPase and vacuole H~+-ATPase under different nutrition and salts stress conditions were analyzed by using qRT-PCR methods. It was found that the transcript of NtHAK1 and NHA1 were significantly stimulated and that of NVP1 was reduced when the K~+ starvation exist in external solution. The results also confirmed that the transcript of K~+ channel gene NKT1, K~+ transporter gene NtHAK1, V-H~+-ATPase G unit gene VAG1 and P-H~+-ATPase gene NHA1 were down regulated when the tobacco was Ca2~+ starved. The transcript of K~+ transporter gene NtHAK1 was induced by excessive Na~+ salinity stress, but that was inhibited when tobacco plant exposed to the solution contained 5 mmol/L NH4~+.
引文
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