Study On Construction Of Over-expression Vector Of Endogenous Phytochrome Genes And Maize Transformation

Maize is the most important food crop in our coutry, which total product covers thirty percents of the world. But, compared with developed coutry,our coutry's unit area of maize is low. How to improve maize yield is the main target in the maize genetic breeding and farming cultivation. Which works to increase the yield are breeding and spreading compact plant types and improving optimum planting density, While, the high planting density would lead to the shortage of lights of maize group, which influence the growth of plants, such as lodging, low maturing rate, diseases and insect pests. In the series of problems, lodging is the most factor to yield. The main reason is that plant shade-avoidance cause plants grow too fast in the high density and stems become feeble and thin, meanwhile, the damage of diseases and insects, attacking of wind and rain influence.Light signals pathways control plants shade-avoidance. Light receptors perceiving environment including three kinds:phytochromeA, B, C in the monocotyledoneae cell. Of the three kinds, both PhyA and PhyB are related to the shade-avoidance. In our research we try to realize over-expression of endogenous phytochrome gene by transgene way, and change the charactors of maize photomorphogenesis by modified light signals pathways, and search to improve maize shade-avoidance and the possibility of yield relevant properties.We chose maize B73 to clone phytochrome gene zmPHY Al and zmPHY A2 by RT-PCR, the whole length of them 3396bp, constructing the expression vectors of them:pTU-A1,pTU-A2. First, new restriction sites had introduced in both ends of the amplified fragments. The fragments were inserted into cloning vector pMD-18T named pMD-18T-A1/A2.Then,pTF101.1 was reformed by inserting promoter Ubiquitin and terminitor T-nos, so obtained the plant expression vector named pTU.On the groundwork, using the fit enzmyes to digest pMD-18T-A1/A2 and pTU, then the digested fragments ligased, constructed monocotyledon expression vector pTU-Al/A2 that was included phytochrome gene A1/A2 cloned from B73.Meanwhile,the vectors included constitutive promoter Ubiquitin cloned from maize, selectable marker gene BAR which promoted by plant constitutive 35S promoter and selectalbe marker gene aadA of E.coli.The expression vectors mediated by Agrobacterium were used to transfer embyronic calli of "18-599R" and "18-599W". After three rounds, With selection gradient of glufosinate medium, resistant calli from "18-599R" transgene-A1 differentiated into 192 regeneration plants,7 Positive plants obtained by PCR amplifiation,respectively, resistant calli from "18-599R" and "18-599W" transgene-A2 differentiated into 60 and 177 regeneration plants,5 Positive plants obtained by PCR amplifiation.Positive plants by PCR amplifiation were pollinated.Owing to absent pollen of some positive plants that PCR detected, we used the non-transgenic and same breed materials'pollen to pollinate the female flower of T0 generation and then obtained T1 generations.T1 generations were detected by PCR. Because T1 generations is not homozygous, they should be identified and selected further.Meanwhile,the positive plant which was just tested by PCR need futher study sueh as molecular hybridization, expression test, molecular mark selection and shade tolerance identification until it is breeding to be steady transgenic breed. In addition to exploration of transferring endogene and improvement of shade tolerance ability,it also help to study the mechanism and function of phytochrome A of the maize.