| Strawberry is to rosaceae strawberry plants’ films, belong to the perennial herbs. Strawberry delicious red is tender, juicy pulp, contains special rich fruit aroma. High nutritional value, contains rich vitamin C, have help digestive effect, at the same time, strawberry can consolidate the gum, pure and fresh and tone, moist throat. Based on the above advantages, strawberry by all alike. Each summer between Jude and July is picking fruit mature season. But because of its skin is extremely thin, high water content, vulnerable to microbial infection and mechanical damage and rot, not high yield, the traditional fresh-keeping technology for the preservation of the strawberry fruit is not a long time, in the transport during storage caused great economic losses. Since the 1980 s, foreign began to research the transgenic plants, our country in transgenic plants of the later start but fast development, at present has had a variety of plants use turn "antisense" ACS or ACO gene technology to delayed fruit mature, prolong storage period,.Related research showed that, in the strawberry fruit mature stage, a large number of expression and ethylene may FaErs1 receptor gene related. Further research FaErs1 gene function will help reveal ethylene receptor and the variant fruit mature step of relation, which for the biological technology to improve the performance of strawberry fruit shipping lay the foundation.Therefore, this experiment with the all star strawberry leaf tissue for materials, plant extracting DNA genome, the PCR amplification get purpose extract. Ers1 purpose extract and build the "antisense" carrier and RNAi expression vector. By agrobacterium mediated into the all-star strawberry plants, for the subsequent establish all-star strawberry genetic transformation system lay the foundation. The main results are as follows:1) According to the genetic sequence of ethylene receptor FaErs1, design a specificity of primers, through the PCR method, from presidents for 2082 bp’s all-star strawberry ethylene receptor gene cloning to in a long for 667 bp of ethylene FaErs1 receptor gene segments. Through the sequencing and use DNAMAN biology software homology, analysis, and the results show that cloned segment GenBank and the registered Chandler-Ers1 homology is as high as 99%. Can show that cloned segment that is all star strawberry ethylene receptor FaErs1 genetic fragments.(2) The design with a pair of SmaⅠ-BamHⅠenzyme cut site specificity primer, reverse cloning all-star strawberry FaErs1 gene fragments, linked to the pBI221 plasmid XbaⅠ-BamHⅠenzyme cut between sites and the forming of middle pBI221 expression vector-preview Ers1. Cut the intermediate expression vector pBI221-the preview Ers1 CaMV 35S promoter and FaErs1 antisense sequence, directional inserted into the resection CaMV 35S the promoter pBI121 plasmid, structured as pBI121-anti-Ers1.(3) Design a pair primer with XbaⅠ-BamHⅠe nzyme cut site specificity , positive cloning all-star strawberry FaErs1 gene fragments, linked to the pBI221 plasmid XbaⅠ-BamHⅠenzyme cut between sites and the forming of middle pBI221 expression vector-preview Ers1. Cut the intermediate expression vector pBI221-preview Ers1 on 35S promoter and sense sequence, it will be inserted into the resection of 35S the promoter pBI121-preview Ers1 plasmid, establishes a RNAi plant expression vector pBI121-Ers1-RNAi.(4) Use of freeze-thaw law will pBI121-preview Ers1, pBI121-RNAi-Ers1 two expression vector to agrobacterium-mediated transformation LBA4404. The specificity of primers transformation of the bacterium fluid PCR amplification, confirmed that express carrier already into agrobacterium-mediated. |
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