Studies On Arabidopsis PLDα1 Mutants Insensitve To Gibberellin

This research is based on the material of Arabidopsis thaliana. The T-DNA was introduced into the wild Arabidopsis thaliana via improved Agrobacterium tumefaciens-mediated transformation system. The insert mutation of T-DNA was gotten. Specific PCR and PCR-Southern blot was carried, which showed that T-DNA fragment has been integrated into the receptor's genome .Because the inserting hot spot is often within the transcript regulation region, we got the mutatant whose inserting spot is within the regulation PLDα1, which cause insensitive to gibberellin and produce many low plants. The expression of PLDαwas affected . So during the late term of growth, ABA was used and getting old of the plant was delayed. The detailed results were as follows:1. The obtaining of T-DNA insertion label community. The T-DNA insertion label community was obtained by using the plasmid PKYL7 reconstrcted and Colombia wild Arabidopsis thaliana via improved Agrobacterium tumefaciens-mediated transformation system. Based on the selection of the plant and PCR , PCR-Southern blot was operated which showed that the T-DNA fragment has been integrated into Arabidopsis thaliana's genome.2. Identification of the T0,T1 representation appraisal of T-DNA label community. By observation and identification of the T0,T1 representation appraisal of T-DNA label community, we found there are several types as followes:(1)Dawlt plant. The decrease of height was variation. The height of some T0 generation was only 7.5cm.No seeds.(2)The plants were precocious on different degree. The life stage of To-01 from planting to mature was only 25 days.(3)The growth ability was different. list stem and weak growing plants were found in T1 generation.(4)There was some percent of white seedling in T0, which died soon after taking out stem. But there was still one plant whose leaf was white with green and seeds were less.(5)Sterile plants were found in T0 generation.3.Arabidopsis breed technique test. Choose three cultivate methods for Arabidopsis breed technique test: transplant method, vermiculite method and 5:5 method.Room culture was performed under the condition of room temperature (26℃about) and artificial illumination (whitefluorescent lamp,16 h illumination,8 h is dark). Under the different raise condition, wild Arabidopsis growed well. But the different raise method, Arabidopsis growth also was different. As for plants after transplanting, the survival rate was lowly, zhe period of little flowers was detention, first flowering highly was short and the number of lotus seed was less.But the life cycle changed little with lengthen protract.4.Gibberelin insensitive mutant screening. Gibberelin screening was performed on transgenic T-DNA label and gibberelin insensitive plants were obtained. Different concentration gibberelin such as 50, 100 and 150PPM were used to the plants, the growth were influenced little. But at the seedling stage, the influence was bigger. Under so large dose gibberelin processing, mutant plants changed little without injury. On the other way, the plants nearly couldn't germinate without the gibberelin processing. All these showed that this mutants were insensitive to gibberelin.5. The comparison test between the mutant and wild Arabidopsis. Mutant plants were drawf and the root grew quite slowly. This difference is remarkable . The growth of vegetal nutrition and reproduction was deficient because of the period'reduction. Under the influence, the flowers of mutant grew slowly. The flowers were fewer and smaller. Even some plants were the male sterility .The reason was the mutant'trophic growth proboly.6. Analysis in Mutant heredity. After the mutant inbred line threegeneration of purification with the wild Ws hybrid, examines F1 and F2generation of high. The result indicated that, F1 generation of highhas the enhancement, but is lower than wildly, calls it middle. But F2appears Gao Zhu, is short, among three separation, the proportion is1: 1: 2, if among will belong to high, will be allowed to present 1: 3separations. This way, this sudden change is altogether the dominantsudden change.7. Falls off the acid treatment analysis. After ABA processes 24 h, thechlorophyll, SOD,POD,the third dial content, the determination indicated, Ws comes under the influence to be bigger, but PLD a 1slightly has the change but not to be obvious.8. Gibberelin insensitive mutant insertion position spot estimation. Through the biological software on-line search and the analysis, andthe pertinent data indicated, T-DNA inserts in structural gene PLDalpha 1 copies the regulative area, causes under the hormone existence, can not very good copy the expression, also is insensitive to the hormone signal response, but causes the PLD alpha 1 expression quantity to reduce, falls off the acid treatment experiment also to beable to provide the certain evidence.