Construction Of Multiple Salt Tolerance Genes Transformation Vectors And Salt Tolerance Difference Of Tobacco Transformation Lines

In order to adapt to the environment of saline soil,the researchers used genetic engineering and biotechnology to construct the vectors of the salt tolerance gene and transfered them into plants to improve the salt tolerance of plants.genetic transformation of many salt-tolerance genes of plant has the advantages of convenient and quick,but multiple salt-tolerance gene were transformed if all can efficiently express and accumulate the genetic effect,to improve plant,salt resistance,it was a problem that in urgent need to clear.In this study,four kinds of salt tolerance vectors were constructed,four vectors were transformed into tobacco and obtain transgenic strains.after the PCR detection and R-T PCR detection and salt resistance test,in order to verify the expression and interactions of salt-tolerance genes in the tobacco,exploring the effect that accumulation of salt-tolerance genes if improve plant,salt resistance,lay the foundation for the application of the genetic transformation of forest trees of multi-gene plant expression vector and the breeding new varieties of salt tolerant genes.the main results were as follows:1、Using the theory of tail with enzyme,through A-T clone on cloning vector,constracting the aim gene ORF,using original enzyme sites disappear principle of after connected with the tail on the enzyme loci of cloning vector and transformation carrier,constantly put purpose gene ORF into carrier,constructed the four kinds of plant expression vectors containing different salt-tolerance genes,which were carriers N27carrying mtl D gene,vector N28 carrying mtl D+gut D genes,vector N29 carrying mtl D+gut D+BADH genes,vector N30 carrying mtl D+gut D+BADH+Sacb genes,the colony was tested by PCR,obtain all purpose fragment,building of four salt-tolerance carriers were successful.2、The tobacco was transformed with the methods of agrobacterium mediated,each carrier obtained five strains,each strain seedling was detected by PCR,purpose gene were detected.3、After different salt stress treatment 30 days,respectively,the transgenic strains of four carriers were detected with fluorescence quantitative PCR,the transcription of aim gene all were detected.under the treatment of water,the transcription abundance of Mtl D gene will increase with the accumulation of gene,except the carrier N29;after 6‰salt stress,transcription abundance increased,the increase proportion of carrier N27 was largest and variation coefficient of the strain was bigger;the transcription abundance of gut D gene increased with the accumulation of gene in water treatment,except the carrier N29,after6‰salt stress,transcription abundance increased,the increase proportion of carrier N28was largest and variation coefficient of the strain of carrier N30 was bigger;the transcription abundance of BADH gene increased with the accumulation of gene in water treatment,after 6‰salt stress,the transcription abundance increased;the transcription abundance of Sacb gene increased,after salt stress.4、Transgenic strains of four kinds of carriers were tested under salt stress,measuring indicators include:seedling growth of various strains,leaves area,chlorophyll content,photosynthetic parameters,chlorophyll fluorescence parameters and biomass index,they can be compared and analyzed,the results showed that:（1）Under the treatment of water,there was no significant difference between plant height of transgenic strains of four carriers and non-transgeneic comparison,show that multi-gene transformation did not significantly influence the growth of tobacco plants;after 6‰salt stress,plant height of transgenic strain of each carrier reduced,the decreased proportion of the plant height of non-transgenic tobacco was largest,each carrier drop ratio was CK>N27>N29>N28>N30.（2）Under the treatment of water,there was no significant difference between leaves area of transgenic strains of four carriers and non-transgeneic comparison;after 6‰salt stress,there was no significant difference between leaves area of transgenic plants of each carrier and water treatment group.（3）Under the treatment of water,the content of chlorophyll a、b of carrier N29strain and non-transgenic plants was highest;after 6‰salt stress,the chlorophyll content of the transgenic plants of each carrier significantly decreased,there was no significant difference between falling proportion of the transgenic plants of each carrier and non-transgeneic.（4）Under 6‰salt stress,the photosynthetic parameters of each carrier strain had decreased,falling rate of the net photosynthetic rate,transpiration rate,stomatal conductance and intercellular CO₂ concentration of non-transgenic strain were significantly greater than transgenic strain,except photosynthetic rate in four carriers,other three parameters decline were no significant difference.（5）Under 6‰salt stress,Fv/Fm and PI values of different carrier transgenic strains had decreased,the difference of the decline rate with the transgenic strains of each carrier and non-transgenic plants was no significant.（6）Under 6‰salt stress,the overground dry weight,underground dry weight,total dry weight of transgenic strains of each carrier had decreased,except carrier N29,there was no significant difference between the falling rate of other carrier all the parts dry weight and non-transgenic strain.5、Many salt resistance indexs by using the methods of subordinate function were comprehensived evaluation and showed that the average salt resistance of transgenic strains of different carrier were higher than non-transgenic plants,the performance of salt resistance of each carrier was the salt resistance of multi-gene plants was greater than the unit price plants,but did not show characteristic of salt resistance absolute increase with accumulation of salt-tolerance genes,the size of the average salt resistance of transgenic strains of different carrier was N29>N28>N30>N27>CK.