Cloning Of Volvariella Volvace Opening Gene And Construction Of Antisense Gene Expression Vector

The development of Volvariella volvacea fruit-body has six stages of the pinhead,the tinny button,the button,the egg,the elongation and the mature.The physiological characteristics of the fruit-body changes obviously in different stages, respiratiry intensity enhanced dramatically especially from the egg stage to the elongation stage.At the same time , the morpha of the fruit-body changes from egg-liking to opening ,which decreases the commercial value of the fruit-body.What makes Volvariella volvacea fruit-body changed so much just in this short time? The cell morpha , internal hormones and the expression of special genes are studied in this paper.1. Cell elongation reduces the rapid growth of fruit-body stipe in the elongation stage We observed cell morpha and measured cell size of different Volvariella volvacea fruit-body stipe and different parts of stipes in the egg and the elongation stages .At the same time we observed and measured the elongation region of fruit-body stipe by stipe labeled.The results showed : fruit-body stipes in egg stage grow and the quantity of the cells increases by cell splitting .The essential factor of the spread of fruit-body stipe has been the elongation of cell on the middle top of stipe since the elongation stage ,in the meanwhile the top cell splitting of stipe plays assistant role.2.Significant relevancy between the change of endogenous hormone and fruit-body growth Determination of the lever change of endogenous hormone of fruit-body stipe at the different development stages shows:there are not significant changes whether growth hormone or senescent hormone.But in elongation stage .growth hormone OA3 and ZR increase dramatically and reach the peak, senescent hormone ABA declines sharply .In mature stage , although the content of GA3 and ZR are lower than those in elongation stage, which remain fairly high .The concentration of ABA reach the lowest lever.The ratio of GA3 and ZR has changed with the concentration change of GA3.the ratio is essential factor which affects the growth of stipe cells.But other growth factors that control sensecence are key effect during senescence from fruit-body to mature..3.Cloning gene fragment expression specific in the opening stageTotal mRNA of fruit-body stipe is regarded as Tester and Driver respectively in egg stage and elongation stage to clone special expression gene fragment of fruit-body stipe in elongation stage by suppression subtraction hybridization.We got a gene fragment and sequence. We did not get the same sequence with our sequence by homology comparision in GENBANK. We took this fragment as template to synthesize primer and have two-dimensional amplification of genome DNA.The amplification with two primer gains target fragment ,at the same time , amplification with single primer get a fragment about 1-2KB.4. Construction of expression vector of sense and antisense gene fragmentTarget gene fragment in plasmid is insolated and cloned with enzyme digest.In the2meanwhile the target expression vector is dealt with end-filling and dephosphorylation. Cloned gene fragment inserts to the polycloning site of PCB vector by connection of blunt end, then transformed E.coli and selected positive clony at random.Plasmids were extracted and sequenced. We verified that we gained cloned vector with reciprocal sequences.