中棉(Gossypium arboreum)光诱导基因Gacab启动子在转基因烟草中的功能缺失分析

Function Deletion Analysis of Light-Induced Gacab Promoter from Gossypium arboreum in Transgenic Tobacco

分离了金华中棉(Gossypiun arboreum var.jinhua)光诱导基因cab 5′上游的调控序列1 009 bp,并对其功能进行了分析,证明获得的这一DNA片段具有驱动光诱导表达的功能。为了进一步分离具有最大转录活性的最小光诱导启动子,根据光诱导表达调控元件所在的位置,构建了Gacab P和197 bp、504 bp、779 bp的5′端缺失体,并将这些缺失体分别与gus(uid A)基因融合,构建植物表达载体。用农杆菌介导法转化烟草,获得转基因烟草。GUS组织化学分析表明,转基因烟草的T1代种子在光下培养时,只有Gacab P驱动gus基因在转基因烟草的叶片表达,其他3个启动子驱动gus基因在转基因烟草的整个植株中均有表达;当转基因烟草的T1代种子在暗中萌发及培养时,GacabP驱动gus基因在转基因烟草中无表达,其他3个启动子驱动gus基因在转基因烟草的整个植株中均有表达。GUS定量分析表明,-504～-1 bp的启动子缺失体启动活性最高,比CaMV35S启动子高0.6倍。上述结果表明只有全长的Gacab启动子具有光诱导和绿色组织特异表达特性,且?504～?1 bp的启动子缺失体启动活性最高。

Genes encoding plant chlorophyll a/b binding proteins （CAB） are a typical group of light-inducible genes. It is well established that cab promoters in plants are light-inducible and tissue-specific. We cloned a 1009-bp promoter sequence of the Gossypium arboreum cab gene and clarified that this promoter （Gacab P） is light inducible. It is also verified that GUS transient expression driven by Gacab P promoter fragment from –504 to –1 bp was significantly higher than that of the CaMV35S promoter. Further work need to be conducted to testify whether this 500 bp segment still maintains light-inducible character. To find the shortest length of light-inducible Gacab promoter with strong transcription activity, the full-length Gacab P （pA） and 5＇ truncations with lengths of 197 bp （pB）, 504 bp （pC）, and 779 bp （pD） were fused with the gus （uid A） gene and ligated into plant expression vectors. All constructs were transformed into Nicotiana tabacum var. NC89 using the Agrobacterium-mediated transformation method. A total of 30 to 35 independent transgenic tobacco lines were generated with each of these constructs. To determine whether the various promoter constructs confer light-regulated expression to gus, F1 progeny seeds of transgenic plants containing different Gacab promoter deletion constructs were geminated in either the dark or light. Ten days later, the seedlings grown in the dark were transferred to the light. GUS histochemical assay showed that gus expression of the pA construct was not detected in the dark, whereas its expression was measurable at green tissue for seedlings grown in the light. gus was expressed throughout seedlings containing pB, pC, or pD grown in either the dark or the light. PlantCARE analysis reveals that some light responsive elements are present between –1009 and –779 bp, including sequences similar to the I-box and the G-box. To analyze the effectiveness of different lengths of the Gacab promoter, GUS expression under the control of the Gacab promoter deletion constructs was examined by fluorometric assays of transgenic tobacco leaf protein extracts. Twenty independent lines for each construct were selected to monitor GUS activity. The average GUS activity is presented for each construct. GUS activity increased when the nucleotides between –1009 and –504 were deleted. Further deletions from –504 to –197 resulted in decreased promoter strength. The highest GUS activity was observed with construct pC, which contained the promoter fragment from –504 to –1 bp. In transgenic plants, the 5＇-deletion Gacab promoter fragments A, B and D had relative activities of 50.2%, 60.0%, and 51.5% respectively （compared with pC）. The –504 to -1 （C） fragment of the Gacab promoter had an activity of 160% compared with CaMV35S. The strength of the other Gacab promoter deletion constructs was similar to CaMV35S. According to these results, we can get conclusion that only the full-length Gacab promoter was light inducible and expressed in a tissue-specific manner, and the promoter fragment from –504 to –1 bp has the highest activity, 0.6-fold higher than that of the CaMV35S promoter.