菠萝AcSERK15’上游区(-499/+258bp)转录活性的研究

Transcriptional activity of the 5' upstream region(-499/+258 bp) of AcSERK1

【目的】探究Ac SERK1启动子的功能,有助于了解Ac SERK1的表达调控模式。【方法】以‘神湾’菠萝(Ananas comosus L.‘Shenwan’)为材料,将Ac SERK1启动子缺失序列与GUS融合,构建植物表达载体,并导入根瘤农杆菌GV3101中。利用农杆菌真空渗透法侵染烟草叶片,并检测GUS活性;利用浸染法转化菠萝胚性愈伤组织获得转基因植株,分析光照、2,4-D和4℃等处理后的GUS表达量。【结果】构建2个Ac SERK1启动子植物缺失表达载体,分别命名为p(-30/+258 bp)和p(-499/+258 bp)。烟草瞬时表达结果显示p(-499/+258 bp)表现出强的GUS活性,p(-30/+258 bp)表现出微弱的GUS活性。q RT-PCR结果表明,光照处理后,GUS表达量降低。相反的,2,4-D和4℃处理转基因菠萝植株后GUS表达量均显著增加。【结论】Ac SERK1启动子-499/-30 bp区段内含有光、生长素和低温响应元件。

【Objective】As a somatic embryogenesis-specific gene, SERK（somatic embryogenesis receptor-like kinase） plays a key role in the development of somatic cells. Analysis of the 5’ upstream region of Ac SERK1 will help us better understand its transcription regulation mechanism. Deletion expression of 5’-flanking region is one of the most significant methods to analyze its promoter function. In this study, deletion expression vectors of the 5’-flanking region fused with a GUS reporter were constructed and then analyzed for the transient expression in tobacco leaves. For further analysis of the promoter function, the stable inherited transgenic pineapple was obtained through using the Agrobacterium-mediated transformation. The putative transgenic plants were analyzed using the molecular biological method. The function of the Ac SERK1 promoter was analyzed by comparing the expression patterns of the reporter gene in transgenic plants which were treated under different conditions.【Methods】The materials used in this experiment were the embryogenic callus of the‘Shenwan’. The Ac SERK1 promoter was truncated at the position of p（-30/＋258 bp） and p（-499/＋258 bp） according to the prediction putative of cis-acting elements and the sites of restriction enzyme which were analyzed by using primer5. These 2 fragments and the p BI121 vector were digested by using Hind Ⅲ and Xba I. After the Ca MV 35 S promoter of p BI121 were replaced by these 2 fragments, the-30/＋258 bp and-499/＋258 bp fragments were fused with the GUS reportergene to construct the plant expression vectors. These recombinant plasmids and p BI121 were transformed into agrobacterium tumefaciens by using the heat-shock method. For the transient expression, the tobacco leaves were infected through Agrobacterium-mediated vacuum infiltration and the GUS activities were examined. After that, the recombinant plasmids were transferred into embryogenic callus by agrobacteriummediated transformation. The kanamycin-resistant regenerated seedling was screened using PCR with the NPTII as the marker gene. To avoid any false positive, the PCR of the NPTII was sequenced and blasted.Then the transgenic plants were treated in light, 5 mg·L- 12,4-D and 4 ℃ at 5 h respectively, and the GUS expression levels were quantitated by real-time quantitative PCR（q RT-PCR） with β-actin as the housekeeping gene.【Results】Two recombinant vectors were verified by enzyme digestion and sequencing. The results showed that the two recombinant vectors were constructed, and were named as p（-30/＋258） and p（-499/＋258）. The tobacco leaves infected with p BI121 were the positive control and the no inoculating tobacco leaves were the negative control. After the tobacco leaves infected with Agrobacterium-mediated vacuum infiltration were cultivated for 36 h under 25 ℃, and the GUS were quantitated by histochemical staining assay. The results of the GUS histochemical staining assay revealed that the activities of GUS were strong in p BI121 and p（-499/＋258） but weak in p（-30/＋258）. This means that p BI121 and p（-499/＋258） had the activity of driving the expression of the GUS gene in tobacco. p（-30/＋258） was a sequence between the ATG and prediction of the transcription start site（TSS）, so the region did not act as a function to drive gene expression. Then p（-30/＋258） and p（-499/＋258） were transformed into the embryogenic callus of the pineapple. Embryogenic callus were cultivated in kanamycin selective mediums after inoculation and co-cultivation. p（-30/＋258） and p（-499/＋258） were obtained from 48 and 56 putative transgenic plants respectively. The DNA was extracted from the putative transgenic plants which were kanamycin resistant. The putative transgenic plants were further detected by PCR with the recombinant plasmids as the positive control, normal DNA as the negative control and H2 O as the blank control. The expected PCR fragment was sequenced. The results show that p（-30/＋258） and p（-499/＋258） were obtained in 14 and29 transgenic plants respectively. In order to analyze the cis-elements of-499/＋258 bp promoter, the expression of GUS gene was analyzed using q RT-PCR with different treatments. The GUS expression of p（-30/＋258） transgenic plants showed no significant changes after light, 5 mg·L-12,4-D and 4 ℃ treatment. The results indicate that- 30/＋258 bp contained the transcription start site but lacked core elements such as TATA-BOX. The GUS expression of p（-499/＋258） transgenic plants declined significantly after light treatment. On the contrary, the GUS expression increased by 3 fold after 5 mg·L-12,4-D treatment compared with the untreated plants. A similar result was found after 4 ℃ treatment, the GUS expression was 1.5 fold greater compared with the 25 ℃ condition. These results reveal that there are some elements that respond to light, 2,4-D and low temperature in the-499/-30 bp region【.Conclusion】Overall,we constructed two recombinant vectors which fused with the GUS reporter gene and finished the transient expression and genetic transformation of the pineapples. The histochemical staining results suggest that there was a transcription start site but no core elements, such as TATA-BOX, in the-30/＋258 bp. 48 and56 putative transgenic plants obtained by kanamycin resistance in p（-30/＋258） and p（-499/＋258） respectively. And in addition, 14 and 29 transgenic plants were detected by PCR and sequenced. The results of the GUS expression showed that the promoter could respond to light, 2,4-D and 4 ℃. In conclusion, there were light- response elements, auxin- response elements and low- temperature- response elements in the-30/＋258 bp fragment.