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Fluorescence Quantitative Analysis and RNAi Inhibition of Pollen Specific Protein CsPSP1from Tea Plant (Camellia Inensis.L.)

Author: GongYing
Tutor: YuMei
School: Anhui Agricultural University
Course: Tea Science
Keywords: Camellia sinensis qPCR antisense expression vector construct RNAidouble-stranded the pollen-tube pathway technology
CLC: S571.1
Type: Master's thesis
Year: 2013
Downloads: 19
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Abstract

Real-time fluorescent quantitative PCR (qPCR)and RNA interfering (RNAi)，generated in1990s，are burgeoning technologies in the field of molecular biology. qPCR isa leap for biological gene expression from qualitative to quantitative. RNAi technology isused for the study of gene function, and it can inhibit gene expression efficiently. Theywould play more and more important roles in biotechnology area in the future.Tea plant is a kind of important cash crop in China, of which the main utilizationvalue was focused on leaves, so there were more researches about quality improvementand increase of the crop. While the studies for tea flowers were relatively few, of courseit’s even more precise about study of inhibiting the reproductive growth to re-promote theeconomic yield of tea plants on gene level. This experiment is to study the related genesfor flower development in tea, and it will do some basic reseach for this blank area.1. Flower buds of four varieties,i.e. Camellia sinensis cv.“Pingyangtezao”,“Wuniuzao”,“Shuchazao”,“Longjing43”, were taken as materials in this experiment, andeach species of bud was classified as the early, middle and late stages according to sizes.The CsPSP1gene expression during different periods of buds was determined by qPCR,the gene expression of the early bud was taken as reference. The results showed that theexpression of CsPSP1gene in middle period buds of Camellia sinensis cv.“Pingyangtezao”,“Wuniuzao”,“Shuchazao”,“Longjing43” were1.8,2.7,2.5,6.5times ofthat in early period buds,respectively; the expression of CsPSP1gene in late period budswere2.5,3.7,3.1,44.2times of that in early period buds, respectively. The results suggestthat CsPSP1gene expression went through an increasing process in flower buds duringdifferent growing periods for each variety,during which the CsPSP1gene expression ofCamellia sinensis cv.“longjing43”increased significantly during three developmentstages,it might be caused by the differences among tea varieties. CsPSP1gene could bededuced to be the late gene during the tea flower developing periods by analysing theexperiment data.2. On the basis of qPCR results,the cDNA fragment of CsPSP1was cloned fromCamellia sinensis cv.“longjing43”by RT-PCR. Meanwhile, according to the sequence ofCsPSP1(DQ887753) in GenBank, a pair of specific primers for the PCR reaction wasdesigned with double enzyme sites. First, CsPSP1fragment was connected to pGEM-7zf(+)vector,then it was inserted into the pBI121reversely.The sequencing result revealed thatthe homologous rate was99.02%, indicating that the vector was successfully constructed. 3. Primers were designed by using pBI121-asCsPSP1as templates. AndasCsPSP1-GUS fragment was amplified, GUS gene was just a part of the entire gene. Thenthe asCsPSP1-GUS was inserted to pGEM-asCsPSP1, to construct the recombinantplasmid pGEM-dsCsPSP1.Finally dsCsPSP1was cut off by endonucleases and insertedinto pBI121to construct the double-stranded expression vector pBI121-dsCsPSP1. Theexperimental results indicated that the length of the double-stranded was1000bp,the400bpGUS among which separate the sense and antisense strands. The RNAi vector wasconstructed successfully by verifying the recombinant plasmid.4. The two kinds of constructed expression vectors were imported into Camelliasinensis cv.“longjing43”by pollen-tube pathway technology respectively and there were450flowers in each treatment. The next year,mature seeds were collected tocultivate.Then,the tea plants grow well and they could be used for the following biologicalidentification

Fluorescence Quantitative Analysis and RNAi Inhibition of Pollen Specific Protein CsPSP1from Tea Plant (Camellia Inensis.L.)

Abstract

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