Heliangine and its encoding gene and application in insect-resisting plant gene engineering
A technology of lectin and Jerusalem artichoke, applied in the direction of genetic engineering, plant genetic improvement, application, etc., can solve the problems of destroying ecological balance, environmental pollution, etc.
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Embodiment 1
[0048] Embodiment 1: Construction of Jerusalem artichoke tuber cDNA library
[0049] The Jerusalem artichoke tuber cDNA library was constructed in the expression-type phage vector λTripIEx2, and the Smart TM cDNA library construction kit, according to the method provided in the manual.
[0050] 1.1 Extraction of RNA
[0051] The extraction of total RNA was carried out according to the method in the literature (Chomezynski and Sacchi, Analytical Boochemistry. 1987, 162: 156-159), with some modifications. Grind 2.0g of tubers (4 weeks of growth) into powder in liquid nitrogen, transfer to a 40ml centrifuge tube, add 5ml of denaturing solution, mix well, add 0.5ml of 2M NaAc (pH4.5), 5ml of water-saturated phenol, 1ml of chloroform After mixing, put it in ice bath for 15 minutes, centrifuge at 10,000g for 10 minutes, add an equal volume of isopropanol to the supernatant, precipitate at -20°C for 1 hour, centrifuge at 10,000g for 10 minutes, discard the supernatant, add 1ml of ...
Embodiment 2
[0090] Example 2: Screening of Jerusalem artichoke tuber cDNA library and acquisition of hta cDNA fragment
[0091] In order to obtain the gene with trypsin inhibitory activity, the present invention measures the inhibitory activity of the induced expression product of the library to trypsin, and performs step-by-step screening of the library on a 96-well plate. take 10 6 A phage from the secondary library was mixed with an appropriate amount of host bacteria (XL1-Blue), LB medium (containing 10mM MgSO 4 , 0.2% maltose, 0.1mM IPTG) were mixed and evenly distributed to each well of a 96-well plate, and after cultivating and multiplying overnight in a shaker at 37°C, according to the assay method for trypsin inhibitor activity (the activity assay of the tool enzyme) : Jiang Chuankui, Shanghai: Shanghai Science and Technology Press, 1980, p105-107), measure the trypsin inhibitory activity of each well, the specific method is as follows: take 50 μ l lysate from each well of 96-we...
Embodiment 3
[0092] Example 3: Expression of hta gene in Escherichia coli and determination of trypsin inhibitory activity
[0093] 3.1 Construction of Escherichia coli expression vector
[0094]The Escherichia coli expression vector pET16 was provided by researcher Huang Hualiang of the Institute of Genetics, Chinese Academy of Sciences. After the plasmid pET16 was digested with Nco I, the large Klenow fragment was added to blunt the end, and then digested with Xho I to recover the vector part. The plasmid pTripHTA was digested with Sma I / Xho I, and the hta fragment was recovered. The hta fragment and the pET16 vector were digested by T 4 DNA ligase was ligated overnight at 16°C, and the ligated product was transformed into Escherichia coli competent cells by electroshock method, and the recombinant pETHTA was obtained by screening. Plasmid extraction, digestion, DNA recovery, and purification were all carried out according to the methods in Molecular Cloning (Molecular Cloning: A Labor...
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