Drought has severely affected the yield and quality of commercial crops.The BRI1 family plays an important role in plant response to drought stress,and BRL3 gene plays an important role in the study of drought in Arab...Drought has severely affected the yield and quality of commercial crops.The BRI1 family plays an important role in plant response to drought stress,and BRL3 gene plays an important role in the study of drought in Arabidopsis thaliana.In this study,NtBRL3 was constructed as a vector and genetically transformed to obtain‘N.Tobacco K326’overexpression of NtBRL3.The enzyme activities of transgenic tobacco and wild-type tobacco were measured and transcriptome and metabolome analyses were performed.The results showed that the antioxidant enzymes of transgenic tobacco were more active under drought conditions,and 85 significantly differentially metabolites and 106 significantly differentially expressed genes were identified in the metabolome and transcriptome analyses,respectively.Transgenic tobacco NtBRL3ox demonstrated an excessive accumulation of droughtrelated metabolites,sugars such as sucrose and maltotetraose,and amino acids such as proline,compared with WT.We discovered drought-related differential genes in the root transcriptome,among which LOX6,RD22,WSD1,CCD8,and UGT were key genes which play an important role in plant response to drought stress.Our results demonstrate that NtBRL3 overexpression in K326 enhances drought resistance in transgenic tobacco.展开更多
Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank...Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank accession number GQ337420）, was isolated from common tobacco （Nicotiana tabacum） leaves by rapid amplification of eDNA ends （RACE）. The NtCDPK12 eDNA is 1 816 bp length and contains an open reading frame （ORF） of 1 461 bp encoding 486 amino acids. Sequence alignments indicated that NtCDPK12 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. The results of real-time quantitative reverse transcription-PCR （qRT- PCR） showed that NtCDPK12 was highly expressed in stems and increased in roots treated with high-salt or subjected to drought stress, which indicates that NtCDPK12 was induced by high-salt and drought stresses.展开更多
Solanesol is an important secondary metabolite in Nicotiana tabacum. Distribution of solanesol in Nicotiana tabacum was investigated by High Performance Liquid Chromatography （HPLC） method. The quantitative distribu...Solanesol is an important secondary metabolite in Nicotiana tabacum. Distribution of solanesol in Nicotiana tabacum was investigated by High Performance Liquid Chromatography （HPLC） method. The quantitative distribution of solanesol in various organs and tissues of N. tabacum showed that solanesol content, obviously different in all organs, was 6.8, 18.3, 27.5, 45.8, and 68.0 times higher in leaves than that in the stalks, flowers, seeds, fruits and roots, respectively. The contents of solanesol in various parts of leaf, stalk and flower were determined. The content of solanesol in top leaf, middle leaf and bottom leaf gradually decreased （6.124, 5.813 and 5.687 mg.g^-1, respectively） and the content of solanesol in various leaf-parts （leaf apex, leaf middle and leaf base） also gradually decreased. The content of solanesol in top stalk was 1.19 times and 1.92 times higher than that in the middle stalk and the bottom stalk, respectively. The content of solanesol in various tissues of stalk （epidermis, cortex and stele） dramatically decreased. The sepal contained higher concentration of solanesol （1.192 mg·g^-1） compared to any other parts in flower. The study will provide the base data for the regulation and control of solanesol, moreover, it will provide the scientific evidences for the rational development and utilization of N. tabacum resources.展开更多
Chalcone synthases （CHS, EC 2.3.1.74） are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synt...Chalcone synthases （CHS, EC 2.3.1.74） are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL （Y14507） gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and important roles in ultraviolet protection and drought tolerance. Our results might play展开更多
A method of preparing exine-detached pollen in Nicotiana tabacum was established. Anthers containing early-middle binucleate pollen were cold-pretreated at 4～6℃ for 7～14 days,and were suspended in 0. 3 mol/L sucros...A method of preparing exine-detached pollen in Nicotiana tabacum was established. Anthers containing early-middle binucleate pollen were cold-pretreated at 4～6℃ for 7～14 days,and were suspended in 0. 3 mol/L sucrose solution for 2 days. During this process,the exine of most pollep grains dehisced. Then they were transferred into an enzyme solution containing 1 % cellulase, 1 % pectinase,0. 1 % pectolyase, I mol/L mannitol, 0. 3 mol/L sorbitol .0. 5 % potassium dextran sulphate and K3 medium macro elemnts. After 15～20 min enzymatic maceration, the exine was detached resulting in the release of exine-detached pollen. Factors affecting preparation of exine-detached pollen were investigated,including cold-pretreatment .osmoticum concentration and enzymes used.展开更多
To further study the function of calcium-dependent protein kinase （CDPK） gene family in common tobacco （Nicotiana tabacum）, it is necessary to isolate more CDPKs from common tobacco and describe the sequence chara...To further study the function of calcium-dependent protein kinase （CDPK） gene family in common tobacco （Nicotiana tabacum）, it is necessary to isolate more CDPKs from common tobacco and describe the sequence characteristics, evolutionary relationship and gene expression. Reverse transcription-PCR （RT-PCR）, rapid amplification of cDNA （RACE） and bioinformatics methods were used to isolate CDPKs from common tobacco. A phylogenetic tree was created using the MEGA4.0 program and expression patterns of the three full-length CDPK genes were studied by RT-PCR. After all aforementioned efforts, we obtained eight additional common tobacco CDPK genes, of which three possessed complete open reading frames （ORFs）. Phylogenetic analysis divided 1 1 full-length Nicotiana CDPK genes into four subfamilies, and two putative common tobacco and Arabidopsis orthologous CDPK genes might correspond to well-conserved functions. Three full-length CDPK genes in common tobacco were detected in all tobacco organs tested, but their expression patterns were significantly different. Eight non-redundant common tobacco CDPK genes were isolated in this study. Along with the previously characterized CDPK genes, at least 15 members of the CDPK family exist in common tobacco. This work establishes a foundation for a genome-wide study of this important gene family in common tobacco.展开更多
This study determined the effects of initial infestation of cowpea seeds （Ife brown variety） with different insect densities （0, 2, 4 and 6 pairs per 50 g seeds） of Callosobruchus maculatus （F.） and evaluated th...This study determined the effects of initial infestation of cowpea seeds （Ife brown variety） with different insect densities （0, 2, 4 and 6 pairs per 50 g seeds） of Callosobruchus maculatus （F.） and evaluated the effects of aqueous leaf extract of Nicotiana tabacum L. on C. maculatus in the laboratory. It was observed that adult beetle population increased significantly （p〈0.05） with increase in insect density. The increase in population of beetles and corresponding weight loss of the seeds in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation, emergence and survival of progeny. Significantly more adults emerged on higher infestation compared to lower and no infestation. In Nigeria, Nicotiana tabacum L. is a locally available plant, with known insecticidal properties. The plant leaf extract was easily extracted with water and confirmed its effectiveness as a protective agent for stored cowpea seeds. Experiment was conducted to assess the effects of aqueous extracts ofN. tabacum at 0, 0.1, 0.2 and 0.3 mL＂ 50 g-1 cowpea seeds on C. maculatus. Data was recorded and showed varying levels of effectiveness against C. maculatus. Result showed that seed appearance was dependent on levels of insect population, while N. tabacum aqueous extract exerted effects on survival of C. maculatus. Aqueous leaf extract of N. tabacum probably contained some insecticidal properties which might have significantly conferred beetle mortality and reduced beetle emergence leading to a decrease in seed weight loss.展开更多
In the present study, five genetically modified herbicide tolerant Nicotiana tabacum cv. TAPM24 plants with a constructed vector pCAMBIA1301a carrying dehalogenase E (dehE) gene were compared with three non-transgenic...In the present study, five genetically modified herbicide tolerant Nicotiana tabacum cv. TAPM24 plants with a constructed vector pCAMBIA1301a carrying dehalogenase E (dehE) gene were compared with three non-transgenic controls using Tto1 retrotransposon specific IRAP markers. dehE gene was originally characterized in Rhizobium sp. and it produced an enzyme which degraded the Dalapon herbicide. IRAP protocol was applied on transgenic and non-transgenic plants to investigate the retrotransposon based genetic variation which may appear during transformation. Polymorphism rates were calculated as 0%-20% from IRAP-PCR products among all plant samples. These results show that transformation of tobacco plant with the dehE gene may cause Tto1 retrotransposon alterations appearing as different band profiles. The findings are expected to contribute to genetic engineering studies to obtain better results and also to understand how transposons contribute to features such as transgenesis. In our knowledge, this is one of the first experimental data of transgenic N. tabacum engineered with dehE gene originated Rhizobium sp. in terms of retrotranposon based variation.展开更多
Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by t...Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. “Red Russian” and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the -Ct value (Ct Actin - Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.展开更多
基金supported by the Science and Technology Project of Guizhou Tobacco Company(2021XM04)the Creation of“Tobacco T-DNA Activation Insertion Mutant Library and Screening of Important Trait Mutants”Project of Guizhou University Talent Introduction(Guizhou University Hezi[2013]50).
文摘Drought has severely affected the yield and quality of commercial crops.The BRI1 family plays an important role in plant response to drought stress,and BRL3 gene plays an important role in the study of drought in Arabidopsis thaliana.In this study,NtBRL3 was constructed as a vector and genetically transformed to obtain‘N.Tobacco K326’overexpression of NtBRL3.The enzyme activities of transgenic tobacco and wild-type tobacco were measured and transcriptome and metabolome analyses were performed.The results showed that the antioxidant enzymes of transgenic tobacco were more active under drought conditions,and 85 significantly differentially metabolites and 106 significantly differentially expressed genes were identified in the metabolome and transcriptome analyses,respectively.Transgenic tobacco NtBRL3ox demonstrated an excessive accumulation of droughtrelated metabolites,sugars such as sucrose and maltotetraose,and amino acids such as proline,compared with WT.We discovered drought-related differential genes in the root transcriptome,among which LOX6,RD22,WSD1,CCD8,and UGT were key genes which play an important role in plant response to drought stress.Our results demonstrate that NtBRL3 overexpression in K326 enhances drought resistance in transgenic tobacco.
基金supported in part by the Special Grand Science and Technology Projects for China National Tobacco Corporation (110200701022,110200902036),Chinathe open subject from the State Key Laboratory of Plant Physiology and Biochemistry (PPB08004)
文摘Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank accession number GQ337420）, was isolated from common tobacco （Nicotiana tabacum） leaves by rapid amplification of eDNA ends （RACE）. The NtCDPK12 eDNA is 1 816 bp length and contains an open reading frame （ORF） of 1 461 bp encoding 486 amino acids. Sequence alignments indicated that NtCDPK12 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. The results of real-time quantitative reverse transcription-PCR （qRT- PCR） showed that NtCDPK12 was highly expressed in stems and increased in roots treated with high-salt or subjected to drought stress, which indicates that NtCDPK12 was induced by high-salt and drought stresses.
基金This paper was supported by Special fund of technological innovation talents in Harbin city, China (No. 2006RFQXN003)
文摘Solanesol is an important secondary metabolite in Nicotiana tabacum. Distribution of solanesol in Nicotiana tabacum was investigated by High Performance Liquid Chromatography （HPLC） method. The quantitative distribution of solanesol in various organs and tissues of N. tabacum showed that solanesol content, obviously different in all organs, was 6.8, 18.3, 27.5, 45.8, and 68.0 times higher in leaves than that in the stalks, flowers, seeds, fruits and roots, respectively. The contents of solanesol in various parts of leaf, stalk and flower were determined. The content of solanesol in top leaf, middle leaf and bottom leaf gradually decreased （6.124, 5.813 and 5.687 mg.g^-1, respectively） and the content of solanesol in various leaf-parts （leaf apex, leaf middle and leaf base） also gradually decreased. The content of solanesol in top stalk was 1.19 times and 1.92 times higher than that in the middle stalk and the bottom stalk, respectively. The content of solanesol in various tissues of stalk （epidermis, cortex and stele） dramatically decreased. The sepal contained higher concentration of solanesol （1.192 mg·g^-1） compared to any other parts in flower. The study will provide the base data for the regulation and control of solanesol, moreover, it will provide the scientific evidences for the rational development and utilization of N. tabacum resources.
基金supported by the Agricultural Science and Technology Innovation Program, China (ASTIP-TRIC01)
文摘Chalcone synthases （CHS, EC 2.3.1.74） are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL （Y14507） gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and important roles in ultraviolet protection and drought tolerance. Our results might play
文摘A method of preparing exine-detached pollen in Nicotiana tabacum was established. Anthers containing early-middle binucleate pollen were cold-pretreated at 4～6℃ for 7～14 days,and were suspended in 0. 3 mol/L sucrose solution for 2 days. During this process,the exine of most pollep grains dehisced. Then they were transferred into an enzyme solution containing 1 % cellulase, 1 % pectinase,0. 1 % pectolyase, I mol/L mannitol, 0. 3 mol/L sorbitol .0. 5 % potassium dextran sulphate and K3 medium macro elemnts. After 15～20 min enzymatic maceration, the exine was detached resulting in the release of exine-detached pollen. Factors affecting preparation of exine-detached pollen were investigated,including cold-pretreatment .osmoticum concentration and enzymes used.
基金supported in part by the Key Scientific and Technological Development Project from State To-bacco Monopoly Bureau,China (110200701021,110200701022)the open subject from State Key Laboratory of Plant Physiology and Biochemistry,China(PPB08004)
文摘To further study the function of calcium-dependent protein kinase （CDPK） gene family in common tobacco （Nicotiana tabacum）, it is necessary to isolate more CDPKs from common tobacco and describe the sequence characteristics, evolutionary relationship and gene expression. Reverse transcription-PCR （RT-PCR）, rapid amplification of cDNA （RACE） and bioinformatics methods were used to isolate CDPKs from common tobacco. A phylogenetic tree was created using the MEGA4.0 program and expression patterns of the three full-length CDPK genes were studied by RT-PCR. After all aforementioned efforts, we obtained eight additional common tobacco CDPK genes, of which three possessed complete open reading frames （ORFs）. Phylogenetic analysis divided 1 1 full-length Nicotiana CDPK genes into four subfamilies, and two putative common tobacco and Arabidopsis orthologous CDPK genes might correspond to well-conserved functions. Three full-length CDPK genes in common tobacco were detected in all tobacco organs tested, but their expression patterns were significantly different. Eight non-redundant common tobacco CDPK genes were isolated in this study. Along with the previously characterized CDPK genes, at least 15 members of the CDPK family exist in common tobacco. This work establishes a foundation for a genome-wide study of this important gene family in common tobacco.
文摘This study determined the effects of initial infestation of cowpea seeds （Ife brown variety） with different insect densities （0, 2, 4 and 6 pairs per 50 g seeds） of Callosobruchus maculatus （F.） and evaluated the effects of aqueous leaf extract of Nicotiana tabacum L. on C. maculatus in the laboratory. It was observed that adult beetle population increased significantly （p〈0.05） with increase in insect density. The increase in population of beetles and corresponding weight loss of the seeds in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation, emergence and survival of progeny. Significantly more adults emerged on higher infestation compared to lower and no infestation. In Nigeria, Nicotiana tabacum L. is a locally available plant, with known insecticidal properties. The plant leaf extract was easily extracted with water and confirmed its effectiveness as a protective agent for stored cowpea seeds. Experiment was conducted to assess the effects of aqueous extracts ofN. tabacum at 0, 0.1, 0.2 and 0.3 mL＂ 50 g-1 cowpea seeds on C. maculatus. Data was recorded and showed varying levels of effectiveness against C. maculatus. Result showed that seed appearance was dependent on levels of insect population, while N. tabacum aqueous extract exerted effects on survival of C. maculatus. Aqueous leaf extract of N. tabacum probably contained some insecticidal properties which might have significantly conferred beetle mortality and reduced beetle emergence leading to a decrease in seed weight loss.
文摘In the present study, five genetically modified herbicide tolerant Nicotiana tabacum cv. TAPM24 plants with a constructed vector pCAMBIA1301a carrying dehalogenase E (dehE) gene were compared with three non-transgenic controls using Tto1 retrotransposon specific IRAP markers. dehE gene was originally characterized in Rhizobium sp. and it produced an enzyme which degraded the Dalapon herbicide. IRAP protocol was applied on transgenic and non-transgenic plants to investigate the retrotransposon based genetic variation which may appear during transformation. Polymorphism rates were calculated as 0%-20% from IRAP-PCR products among all plant samples. These results show that transformation of tobacco plant with the dehE gene may cause Tto1 retrotransposon alterations appearing as different band profiles. The findings are expected to contribute to genetic engineering studies to obtain better results and also to understand how transposons contribute to features such as transgenesis. In our knowledge, this is one of the first experimental data of transgenic N. tabacum engineered with dehE gene originated Rhizobium sp. in terms of retrotranposon based variation.
文摘Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. “Red Russian” and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the -Ct value (Ct Actin - Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.
