| Tubulin plays an important role in the life activities of cells.Apart from participating in the maintenance of cell morphogenesis,intracellular substance transport,biological movement,and cell division and other physiological functions,tubulin also participates in plants responding to a variety of adversity stresses.In this study,Gramineae Arundo JUNCAO Lvzhou No.1 was used as the research object,plant tissue culture and genetic engineering techniques were used to transfer the Tubulin gene αtubulin(tua)to Lvzhou No.1,and initially established the Lvzhou No.1 tissue culture system and genetic transformation system and access to transgenic Lvzhou NO.1.The aim was to cultivate transgenic Lvzhou NO.1 germpalsm with strong cold resistance.The main results were obtained as follows:First,by designing a four-factors and five-levels of orthogonal test,it was found that the medium(MS+1.5 mg/L 2,4-D+1.0 g/L PVP)had the best effect on inducing callus,and the rate of callus was up to 79%.Callus quality was also the best.Second,by designing a three-factor three-level central composite test,the medium(MS+2.0 mg/L 6-BA+0.3 mg/L NAA+1.0 mg/L KT)was selected,the effect of inducing adventitious buds was best,and the medium could directly promote adventitious buds rooting.Third,agrobacterium-positive clones were cultured under the conditions(YEP+100 mg/L Kan+25 mg/L Rif+25 mg/L Str),and verified by PCR amplification and sequencing.The similarity of tua gene and saccharum hybrid cultivar GT28 tubulin alpha-5(tua5)mRNA complete sequence were found to be 99%.When 150 mg/L Tim was added to the YEP liquid medium,the growth of agrobacterium could be completely inhibited within 15 days.The survival rate and rooting rate of Lvzhou No.1 tissue culture seedlings gradually decreased as the concentration of PPT increasing.When the concentration of PPT in the medium is 5 mg/L,rooting can be completely inhibited,the lethality of the tissue culture seedling is 100%.The infection concentration was 0.6A,infection for 40 minutes,co-cultured for 5 days,and the highest conversion rate was 27.08%.Forth,five positive plants were selected randomly to analyze the transcription level of α-tubulin gene in the leaves of Lvzhou No.1 by qRTPCR technology.The were significant differences in the transcription level of different plants.The highest expression reached 27,330 copies/μl,the lowest expression was only 2,021 copies/μl.In summary,this study initially established the tissue culture system and genetic transformation system of JUNCAO Lvzhou No.1,which would provided theoretical guidance for molecular breeding and industrialization of JUNCAO Lvzhou No.1. |
