Construction Of Regeneration System And Clone Identification Of DsEXLA2 In Dendrocalamus Sinicus

Bamboo is a potential renewable biomass resource due to its fast growth,early timber production,strong adaptability,high yield,easy planting,wide distribution and easy processing.Since bamboos lack cambium and are rich in secondary metabolites,the establishment of regeneration system of bamboos is a difficult point in botany research.Currently,only a small number of bamboos have established a complete regeneration system,resulting in a great limitation of their genetic improvement.Dendrocalamus sinicus is the largest bamboo species in the world,and it grows very fast.It is a typical example of clumped bamboo in the hot areas of southern China.As a cell wall loosen protein,expansin plays a key role in regulating seed germination,root formation,leaf development,stem and node elongation,stomatal movement,flower development,fruit development and maturation.However,the function of expansin gene in the rapid growth of D.sinicus stem remains unclear.On the basis of previous transcriptome data discovery expansin-like A2（EXLA2）genes expressed in different internode significant difference.In this study,a regeneration system of DsEXLA2 was constructed by tissue culture.In addition,the sequence,spatial and temporal expression pattern,subcellular localization and transgenic Arabidopsis thaliana were studied by RACE cloning,overexpression and RNA interference vector genetic transformation..The main results are as follows:1.An efficient system for callus induction and plantlet regeneration was established using hypocotyls of D.sinicus.Box-Behnken model showed that the effects of 6-BA,citric acid and2,4-D on callus induction rate decreased gradually,and the optimal concentrations were 2.10mg/L,420 mg/L and 3.10 mg/L,respectively.Under these conditions,the predicted callus induction rate was as high as 89.06%,and the verified callus induction rate in this experiment was 88.87%.The highest frequency of callus formation was on Murashige and Skoog（MS）medium containing 30 g/L sucrose,6 g/L agar,500 mg/L casein hydrolysate（CH）,500 mg/L proline（Pro）,500 mg/L glutamine（Gln）,2.10 mg/L 6-BA,420 mg/L citric acid and 3.10 mg/L2,4-D.Comparison of different browning inhibitors citric acid,ascorbic acid and activated carbon showed that 400 mg/L citric acid had the best inhibition effect on callus browning.After further differentiation and rooting,the rooting rate and transplanting seedling survival rate reached 91%and 93%,respectively.2.The full length of EXLA2 gene was cloned from the stem of D.sinensis by RACE method,and was named DsEXLA2 according to the homology alignment results.The full length of c DNA and g DNA sequences of the gene were 1025 bp and 1746 bp,respectively,encoding 276 amino acids with a relative molecular weight of 29.62 k Da.It was a stable hydrophobic protein containing a signal peptide and a transmembrane region.The DsEXLA2gene contains two conserved domains,DPBB＿1 and Pollen＿allerg＿1,and the CDRC motif,cysteine and tryptophan are highly conserved among different species.The gene is evolutionarily conserved and contains 5 exons and 4 introns.It is closely related to Oryza sativa,Setaria italica and Panicum hallii of gramineae.3.The expression level analysis of DsEXLA2 in different internodes and different tissues showed that the expression of DsEXLA2 increased from the top of internode to the base of internode in one-year old stem.The seeds,roots,stems and leaves of seedlings,as well as stems and leaves of two-years old plant were selected for gene expression analysis,and it was found that the expression of DsEXLA2 was the highest in seeds and lowest in leaves of two-years old plant.The gene expression was followed by seeds>the leaves of seedlings>the stems of seedlings>the roots of seedlings>the leaves of two-years old plant>the stems of two-years old plant.Subcellular localization showed that DsEXLA2 protein was localized to the cell wall.4.The overexpression vector p CAMBIA1301-35S-NOS-DsEXLA2 and RNA interference vector p TCK303-DsEXLA2 were constructed.Through agrobacterium-mediated transformation of A.thaliopsis,and identified by PCR and q RT-PCR,9 overexpression lines and 5interference lines were screened respectively,and the homozygous T₃ generation was obtained.5.Compared with the wild type,the transgenic plants with overexpression of DsEXLA2had higher plant height,thicker stem,larger leaves,more epidermal hairs,and larger stomata.The results showed that DsEXLA2 gene could promote the growth and development of stem and leaf.In addition,the content of cellulose in the stems of the transgenic plants overexpressed with DsEXLA2 was increased compared with that of the wild type,and the cell wall of the transgenic plants overexpressed with DsEXLA2 was significantly thickened by paraffin section.These results indicate that DsEXLA2 gene is involved in the cell wall related growth and development regulation.In this study,a stable and efficient regeneration system was established for the first time through the comprehensive optimization of callus induction,differentiation,rooting and browning inhibitors.In addition,the DsEXLA2 was cloned and the genetic transformation was carried out in A.thaliana.The results of the study will facilitate to reveal the mechanism of DsEXLA2 in the growth and development of the stem,promote the mining and utilization of the excellent traits of this important bamboo species,and provide reference for the molecular breeding of other bamboo resources.