| About 20% of the arable land area in the world is affected by soil salinization and drought stress.These two stresses are the main abiotic stress factors for crop yield reduction.Xinjiang is the region with the most extensive salinization area and the highest soil salt accumulation in China,accounting for 22.0% of the country's saline-alkali land area.Tomato is one of the important economic crops in Xinjiang.With the development of high-intensity water and soil resources in the arid regions of Xinjiang in recent years,the balance of traditional irrigation and drainage salts has been destroyed,and the increasing soil salinization will cause crop yield and quality to decline.Therefore,while improving soil salinization,it is also necessary to improve the salt tolerance of processed tomatoes,and to cultivate new salt tolerance varieties for processing tomatoes in order to mitigate the effects of salt stress on processed tomatoes.The processing of tomatoes is a moderately salt-tolerant material and the breeding of new salt-tolerant varieties is one of the aims and directions of tomato resistance breeding.Therefore,this experiment used ethyl methanesulfonate(EMS)mutagenesis to process the tomato "JW9" material to screen for salt-tolerant mutants,use the RNA-seq sequencing technology to analyze the salt-tolerant genes and clone and construct the expression vector in order to utilize the transgenic technology to transform Arabidopsis,so as to obtain new salt-tolerant genes after verification.The results of the study are as follows:1.Observe the type of mutation in field cultivation: Using EMS(3.0%)mutagenesis to treated 1500 processing tomato seeds,and they were sown for 10 h and sowed in trays.140water-treated tomato seeds were treated with fresh water for 10 h as contrast.At the seedling stage,725 EMS-treated materials survived and all the contrasts survived.After the fruits matured,single plants were planted.The treatment group survived and received 592 strains.The contrast group survived.The treated plants were also found the abnormalities with yellow seedlings,lack of cotyledons,no growth points,purple leaves,pale green leaves and short seedlings.When they have four-leaf and one-heart,they can be transplanted into the field,the morphological observations revealed the following types of variation: high plants,low plants,stems non-branching,plants non-flowering,stems branching,male sterility,no growth point,late maturing and thick stems;leaf color was light green,leaf curling,large leaves,narrow and long leaves and leaf shortage;the total variation rate of milky ridges,cracked tops,gourd fruit,streaked fruit and deformed fruits and so on.The total variation type of M1 generation tomato is 26 species,the total variation number is 133,and the variation rate is 8.87%.The beneficial mutation is that the plant is tall,the photosynthetic capacity of the plant is increased,the amount of fruit is large,the stem is thicker,the resistance to toppling is strong,the number of branches of the plant is increasing,and the result is more.Pollen abortion resulted in male sterility of tomato,which provided materials for heterosis.2.Using PCR-SSCP technology to screen salt-tolerant mutants: the 592 treatment groups and all the contrast groups that survived in the field were sampled.DNA was extracted from592 samples and contrast samples,and mutants were screened for the salt stress-related gene primers by using SSCP molecular marker technique 50.The results showed that 22 pairs of primers with stable bands were screened out.Of the 592 plants that were mutagenized,17 bands were different from the contrast materials.The polymorphism detection rate was 2.87%,and 22 pairs of primers were detected.A total of 23 polymorphic sites were detected with 6pairs of primers.The primers screened from the SSCP technique accounted for 27.27% of all primers that could amplify the band and accounted for 12% of all screened primers.3.RNA-seq data analysis of candidate genes: The analysis of the RNA-seq sequencing results of 16 salt-tolerant mutant plants selected in the previous period in the laboratory found that 4367 genes were selected as the significant differentially expressed genes(DEGs)among samples,among which 2606 genes were up-regulate,and own-regulated genes were 1761.These significant differentially expressed genes function mainly involve secondary metabolism,metabolic regulation,hormone metabolism,signal transduction and plant pathogen interaction.The q RT-PCR assay showed that the expression of 10 randomly selected DEGs was consistent with that of the expression profiling.Through GO and KEGG pathway enrichment analysis,from the genes associated with salt-promoting serine/threonine protein kinase and ATPase enzyme synthesis,five significant differentially expressed genes were found including Solyc08g066270.1 and Solyc03g083420.2 and Solyc10g018530.1,Solyc09g082890.1 and Solyc02g014190.2,and they may be salt-tolerant genes.4.Salt-tolerant candidate gene clone: Genes were cloned from the candidate genes.The m RNA sequence and size of the candidate genes were identified by RNA-seq sequencing.After homologous sequences were compared using Blast,the gene core sequences were identified using MEGA6 and primers were designed using Primer6 primers,and at the same time,the enzyme cut site sequence were added.16 strains of salt-tolerant mutants were used to extract c DNAs synthesized from total RNA.The target bands were amplified by PCR and cloned into p MD19-T vector for sequence verification.The successfully tested genes were subcloned into the plant overexpression vector p CAMBIA2300 to construct the appropriate recombinant was verified by double digestion and sent to sequencing.It was found that only the Solyc02g094400.2 gene overexpression vector was successfully constructed. |
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