CN102220371A - Application of tomato TFT1 gene to improving salt tolerance of crops - Google Patents
Application of tomato TFT1 gene to improving salt tolerance of crops Download PDFInfo
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Abstract
The invention relates to the application of tomato TFT1 gene to improving the salt tolerance of crops, comprising the following steps of: obtaining the DNA sequence of TFT1, cloning the encoding area of TFT1 gene in a tomato body through the PCR method, connecting the encoding area to a modified vector pBI121 after sequencing identification (see figure 1), and transforming targeted crops through the floral dip method. The crops in which TFT1 is transplanted are all along endowed with higher APX activity no matter under a normal growth condition or salt stress condition, thereby indicating that the TFT1 has the function of improving the salt tolerance of crops.
Description
Technical field
The invention belongs to gene engineering technology field, relate to the application of tomato TFT1 gene in improving salt tolerance of crop.
Background technology
The soil salinization is one of major reason of China's grain drop in production.The approach and the method for improvement salinate fields are a lot, and Recent study shows that the genetic potential that improves the anti-soil salinization of plant also is one of effective means.In recent years, many plant 14-3-3 albumen that studies show that are resisted in the salt stress process and may be played a leading role the regulation and control plant.People utilize transgenic plant and mutant doing many work for material aspect the plant salt tolerance mechanism, but 14-3-3 albumen aspect effect research less.Although 14-3-3 albumen is resisted in the salt stress process and may be played a leading role the regulation and control plant, the direct evidence of this respect is less.Tomato is the very strong crop of adaptability, and cultivation is all arranged from the north polar circle to the equator.Although tomato is very strong to various weather condition adaptability, coercing the influence of output of the poor environment factor is still very big, and salt stress is one of these poor environments and the yield and quality that has a strong impact on tomato.Therefore, the salt resistance ability of raising tomato is significant.14-3-3 protein gene family has 12 members in the tomato body, and TFT1 is number one member wherein.Therefore, this research and establishment the transgenic arabidopsis of overexpression TFT1, to change TFT1 Arabidopis thaliana and wild-type contrast Arabidopis thaliana make comparisons (Columbia), TFT1 role and functional effect in the plant salt tolerance process have been studied, can provide applicable gene for ability and the salinification of improvement environment that improves plant salt tolerance by molecular biology method, also lay further research basis for the salt resistance ability that improves tomato by the molecular breeding means.
Summary of the invention
Technical problem: the present invention is directed to the present situation of the soil salinization, improve the genetic potential of the anti-soil salinization of plant, thereby improve salt tolerance of crop by tomato TFT1 gene.
Technical scheme: the application of tomato TFT1 gene in improving salt tolerance of crop.Obtain the dna sequence dna of TFT1, with the PCR means from the tomato vivo clone coding region of TFT1 gene, after order-checking is identified, be connected to transformed carrier pBI121, see Fig. 1, transform target crop to dip in colored method again.
Beneficial effect: no matter under the normal growth condition or under the condition at salt stress, change the TFT1 crop and all have higher APX activity, show that TFT1 has the effect that improves the salt tolerance of crop energy.
Description of drawings
The carrier of Fig. 1 arabidopsis thaliana transformation;
The space structure of Fig. 2 TFT1;
At-L23a and TFT1 expression of gene in Fig. 3 wild-type and the commentaries on classics TFT1 Arabidopis thaliana body;
Under Fig. 4 salt stress, H in wild-type and the commentaries on classics TFT1 Arabidopis thaliana body
2O
2With MDA content; Article three, data are one group, are respectively from left to right: WT/L2/L6.
Under Fig. 5 salt stress, wild-type and commentaries on classics TFT1 Arabidopis thaliana body endoenzyme activity change.Article three, data are one group, are respectively from left to right: WT/L2/L6.
Embodiment
Materials and methods
Vegetable material, growth conditions, salt stress are handled
The means that we cultivate tomato are water planting, and nutritive medium is 1/5Hoagland.We utilize MS plate (0.8%agar and 3%sucrose) to cultivate Arabidopis thaliana.The processing means are 0,80,100,115,130 and 150mM NaCl.We do sprouting test with about 100 Arabidopis thaliana seed, broadcast the MS plate and count after 5 days, and passing kind of skin with radicle is sprouting.For growth test, the Arabidopis thaliana that we get the growth unanimity of normal sprouting after 5 days is a material, and it was moved on to the growth of MS plate after 10 days, gets Arabidopis thaliana plant (seedling age 15 days) after liquid nitrogen is fixing, is stored in-80 ℃ of refrigerators with usefulness to be analyzed.Each test triplicate is got 10 Arabidopis thaliana plant at every turn.In addition, each test repeats (the biological repetition) at least twice.
Make up transgenic arabidopsis
Extract tomato RNA and reverse transcription and be cDNA (Xu WF and Shi WM.2006.Expression of profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots:analysis by real-time RT-PCR.Annals of Botany, 98:965-974.).We obtain the dna sequence dna of TFT1 from the website of NBCI, with the PCR means from the tomato vivo clone coding region of TFT1 gene, after order-checking is identified, be connected to transformed carrier pBI121, see Fig. 1.The method of all molecule manipulations is seen " molecular cloning experiment guide " third edition (Sambrook et al., 1989).We are to dip in colored method arabidopsis thaliana transformation (Clough SJ, Bent AF.1998.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.The Plant Journal 16:735-743.).Through after the series of selection, cultivation, evaluation, we are that material carries out analysis of experiments with isozygotying of transgenosis T3 generation at last.Real-time RT-PCR; Real-time RT-PCR method see (Xu WF and Shi WM.2006.Expression of profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots:analysis by real-time RT-PCR.Annals of Botany, 98:965-974.).
The primer of table 1 Arabidopis thaliana Real-time RT-PCR
The measurement of the prediction of protein structure and root parameter
We have predicted the proteic space structure of TFT1 with SWISS-MODEL (http://swissmodel.expasy.org).We with the root system scanner measure total root long (WinRHIZO, Regent, Canada).
Enzyme activity determination
The measuring method of APX, CAT, GPX is with Aravind and Prasad (Aravind P, Bi ℃ of hemistry 43:107-116. of Prasad MNV.2005.Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism.PlantPhysiology and).
The mensuration and the statistical study of conventional physical signs
Dry weight, cations, chlorophyll, protein, H
2O
2Mensuration with MDA.Use SPSS13.0Duncan ' s Multiple Range Test to carry out statistical study (P＜0.05).
The result
The space structure of TFT1 is seen Fig. 2.
TFT1 is one of tomato 14-3-3 protein family member, resists in the soil environment stress process and may play an important role the regulation and control plant.Some reports show that the TFT1 expression of gene is subjected to (Wang et al., 2001 of inducing of potassium deficiency, iron deficiency and nitrogen; Wang et al., 2002).We also show under the salt stress at result of study, TFT1 genetic expression in the tomato root raise and in 12 members of tomato 14-3-3 family expression amount the highest (Xu WF and Shi WM.2006.Expression of profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots:analysis by real-time RT-PCR.Annals of Botany, 98:965-974.).We have predicted the proteic structure of TFT1 (Fig. 2) with SWISS-MODEL, and space configuration has the typical anchor shape of plant 14-3-3 albumen structure.
Change the evaluation and the selection of TFT1 Arabidopis thaliana strain system:
We have analyzed wild-type contrast Arabidopis thaliana (Columbia) and have changeed intravital At-L23a of TFT1 Arabidopis thaliana and TFT1 expression of gene (Fig. 3).At-L23a is in wild-type contrast (WT) and change TFT1 Arabidopis thaliana strain system (L1-L6) expression indifference, and this method and Real-time RT-PCR that shows that we take a sample tests system accurately and reliably.We studies show that TFT1 does not have expression in wild-type Arabidopis thaliana body, and in TFT1 Arabidopis thaliana strain system (L1-L6) body expression are arranged all, and this shows that the certain overexpression of TFT1 is in the body of Arabidopis thaliana.According to the expression of results of TFT1, we have selected following strain to be: L2 (the TFT1 expression amount is minimum), L3 (the TFT1 expression amount is the highest), three strains of L6 (the TFT1 expression amount is medium) are research experiment.
Change the analysis of TFT1 arabidopsis thaliana salt-tolerance
We have analyzed (0,80,100,115,130,150mM NaCl) under the different concns salt stress, wild-type contrast and commentaries on classics TFT1 Arabidopis thaliana strain system (L2, L3, L6) intravital germination rate, dry weight, chlorophyll content, total root long (table 2).Result of study shows: 1) (115,130,150mM NaCl) under the salt stress, and change the TFT1 Arabidopis thaliana and be higher germination rate than the wild-type contrast; 2) (100,115,130mMNaCl), commentaries on classics TFT1 Arabidopis thaliana is kept higher dry matter weight than the wild-type contrast under the salt stress; 3) under the salt stress (80,100,115mMNaCl) and the wild-type contrast ratio, it is higher to change the intravital chlorophyll content of TFT1 Arabidopis thaliana; 4) (80,100,115,130,150mM NaCl) under the salt stress are changeed the total root of TFT1 Arabidopis thaliana and are longer than the wild-type contrast.These results show and the wild-type contrast ratio that it is higher that commentaries on classics TFT1 arabidopsis thaliana salt-tolerance is coerced ability.Our result also shows and the wild-type contrast ratio, and the TFT1 arabidopsis thaliana salt-tolerance ability of coercing not only is embodied in the sprouting stage and also finds expression in growth and development process (dry weight, chlorophyll content, total root are long).In addition, under the 115mMNaCl salt stress, the index of all mensuration all is considerable change, and the growth phenotype is (Fig. 4) also clearly, so we select 115mM NaCl (10 days) to be the concentration of treatment of further analysis of experiments.
Under table 2 salt stress, the mensuration of wild-type and commentaries on classics TFT1 Arabidopis thaliana growth indexes
Change TFT1 Arabidopis thaliana strain system and wild-type contrast (Columbia) intravital cations
We have analyzed commentaries on classics TFT1 Arabidopis thaliana strain system (L2 and L6) and the intravital cations of wild-type Arabidopis thaliana (table 3).Result's demonstration, after 115mM NaCl concentration is handled 10 days, Na in Arabidopis thaliana (L2, L6 and the wild-type) body
+Concentration raises, and K
+And Ca
2+Concentration is on a declining curve, Mg
2+Change in concentration is little.Further no matter analysis revealed is still coercing under the processing at 115mM NaCl under the normal growth condition, changes TFT1 Arabidopis thaliana strain system and the intravital cations (Na of wild-type Arabidopis thaliana
+, K
+, Ca
2+And Mg
2+) difference is not obvious.
Under table 3 salt stress, wild-type and the intravital cations of commentaries on classics TFT1 Arabidopis thaliana
Wild-type contrast and the intravital H of commentaries on classics TFT1 Arabidopis thaliana
2O
2With MDA content
We have analyzed commentaries on classics TFT1 Arabidopis thaliana strain system (L2 and L6) and wild-type contrast (Columbia) intravital H
2O
2With MDA content (Fig. 4).The result shows, under normal growth conditions, changes TFT1 Arabidopis thaliana and wild-type and contrasts intravital H
2O
2With MDA concentration no significant difference.After 115mM NaCl concentration is handled 10 days, change TFT1 Arabidopis thaliana and the intravital H of wild-type
2O
2All in rising trend with MDA concentration.After 115mM NaCl concentration is handled 10 days, compare simultaneously, change the intravital H of TFT1 Arabidopis thaliana with wild-type
2O
2Lower with MDA concentration, obvious difference (P＜0.05).These results show under salt stress, compare with wild-type contrast (Columbia), and it is lighter that commentaries on classics TFT1 Arabidopis thaliana is subjected to the oxidative stress injury.
Changeing the intravital enzymic activity of TFT1 Arabidopis thaliana and wild-type contrast (Columbia) changes
We analyzed antioxidase in commentaries on classics TFT1 Arabidopis thaliana strain system (L2 and L6) and wild-type contrast (Columbia) body (CAT, GPX, activity APX) (Fig. 5), these 3 kinds of enzymes are direct removing H in the plant materials
2O
2Main enzyme.The result shows that under the normal growth condition, it is not obvious to change interior CAT of TFT1 Arabidopis thaliana and wild-type body and GPX activity difference, and the APX activity is obviously higher.After 115mM NaCl concentration was handled 10 days, it was active on a declining curve but difference is not obvious to change TFT1 Arabidopis thaliana and the intravital CAT of wild-type and GPX, and compares with wild-type, keeps higher APX activity (P＜0.05) in the commentaries on classics TFT1 Arabidopis thaliana body.These results show under salt stress, compare with wild-type contrast (Columbia), change in the TFT1 Arabidopis thaliana body and can keep higher APX activity.
Conclusion
It is irrelevant with the interior ion homeostasis of body to change TFT1 arabidopsis thaliana salt-tolerance mechanism
Our result shows, compares with wild-type contrast (Columbia), changes the TFT1 Arabidopis thaliana and has the phenotype of salt tolerant.Some reports show that salt stress has caused the intravital nutrient ions imbalance of plant, so one of plant important mechanisms that to keep intravital ion homeostasis also be plant salt tolerance (Zhao et al, 2006).Table 3 is the result show, no matter still coerces down at 115mM NaCl under the normal growth condition, and commentaries on classics TFT1 Arabidopis thaliana and the intravital cations difference of wild-type Arabidopis thaliana are not obvious.These results show that the mechanism and the interior ion homeostasis of body that change the TFT1 arabidopsis thaliana salt-tolerance are irrelevant.
Change the TFT1 arabidopsis thaliana salt-tolerance mechanism can by vivo oxidation coerce extent of injury alleviate explain
As everyone knows, the intravital anti-oxidant system of coercing of plant is one of mechanism of plant salt tolerance.Therefore, we study changes TFT1 Arabidopis thaliana and wild-type contrast (Columbia) intravital oxidative stress level under the salt stress.As shown in Figure 4, under salt stress, compare with wild-type, changeing TFT1 Arabidopis thaliana vivo oxidation, to coerce extent of injury lighter, the H that is produced by salt stress in the body
2O
2Content is lower.These results show under salt stress, change the TFT1 arabidopsis thaliana salt-tolerance mechanism can by vivo oxidation coerce extent of injury alleviate explain.
To sum up, we utilize commentaries on classics TFT1 Arabidopis thaliana and wild-type contrast to reach a conclusion for material experiment, compare with wild-type, no matter under the normal growth condition or under the condition at salt stress, change the TFT1 Arabidopis thaliana and all have higher APX activity.These results show that TFT1 has the effect that improves the salt tolerance of crop energy.
SEQUENCE?LISTING
＜110〉Nanjing Soil Inst., Chinese Academy of Sciences
＜120〉application of tomato TFT1 gene in improving salt tolerance of crop
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Claims (2)
1. the application of tomato TFT1 gene in improving salt tolerance of crop.
2. the application of tomato TFT1 gene in improving salt tolerance of crop, it is characterized in that obtaining the dna sequence dna of TFT1, with the PCR means from the tomato vivo clone coding region of TFT1 gene, after order-checking is identified, be connected to carrier pBI121, transform target crop to dip in colored method again.
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Cited By (1)
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|---|---|---|---|---|
| CN102766638A (en) * | 2012-07-09 | 2012-11-07 | 中国科学院南京土壤研究所 | Application of tomato TFT6 and TFT7 transgenes in enhancing phosphorus-deficient soil environment endurance of plants |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386645A (en) * | 2008-10-29 | 2009-03-18 | 中国农业科学院作物科学研究所 | Plant salt tolerant protein and encoding gene and application thereof |
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2011
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386645A (en) * | 2008-10-29 | 2009-03-18 | 中国农业科学院作物科学研究所 | Plant salt tolerant protein and encoding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《Annals of Botany》 20061231 WEI FENG XU ET AL. Expression Profiling of the 14-3-3 Gene Family in Response to Salt Stress and Potassium and Iron Deficiencies in Young Tomato (Solanum lycopersicum) Roots: Analysis by Real-time RT-PCR 965-974 1-2 第98卷, * |
| 《DNA Research》 20061231 Fang Chen et al. The Rice 14-3-3 Gene Family and its Involvement in Responses to Biotic and Abiotic Stress 53-63 1-2 第13卷, * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766638A (en) * | 2012-07-09 | 2012-11-07 | 中国科学院南京土壤研究所 | Application of tomato TFT6 and TFT7 transgenes in enhancing phosphorus-deficient soil environment endurance of plants |
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Application publication date: 20111019 |