Clone of novel gene sequence expressed and suppressed in period of hibernation in young follage on the top end of tea plant
A technology of young buds and hibernation, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve problems that have not been considered
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Embodiment 1
[0141] RNA isolation, digestion of RNA using DNase 1, RNA quantification and gel electrophoresis
[0142] In order to ensure that high-quality ribonucleic acid (hereinafter referred to as RNA) is obtained from the ND and D shoots of tea plants, the RNase (RNeasy) plant kit (purchased from M / s. Qiagen in Germany) is used. Isolate RNA according to the manufacturer's instructions. The RNA was quantified by measuring the absorbance at 260nm, and the purity was monitored by calculating the ratio of the absorbance measured at 260nm and 280nm. It is generally believed that the value at 260 / 280nm>1.8 is required for the purpose of this study. The equations used to calculate RNA concentration and yield are as follows:
[0143] Concentration of RNA (μg / ml) = A 260 (Absorbance at 260nm)×40×dilution factor
[0144] Total yield (μg) = concentration × volume of spare RNA sample
[0145] In order to test the integrity of RNA, use 15.5μl of M1 solution (2μl of 5X MOPS buffer, 3.5μl formaldehyde a...
Embodiment 2
[0148] Reverse transcription (hereinafter referred to as RT) to convert mRNA into complementary DNA (hereinafter referred to as cDNA)
[0149] Using a known enzyme as a reverse transcriptase, 0.2 μg of DNA-free RNA from dormant and non-dormant samples was subjected to reverse transcription in the separation reaction. Use 0.2μM T 11 M primer (T 11 M in M can be T 11 A, T 11 C or T 11 G), 20μM dNTP, RNA and RT buffer [25mM Tris-Cl (pH8.3), 37.6mM KCl, 1.5mM MgCl 2 , And 5mM DTT] complete the reaction. In the present invention, dNTP refers to deoxyadenosine triphosphate (hereinafter referred to as dATP), deoxyguanosine triphosphate (hereinafter referred to as dGTP), deoxycytidine triphosphate (hereinafter referred to as dCTP) and deoxythymidine triphosphate (hereinafter referred to as dTTP) ). The three RT response settings correspond to T 11 M primer for each RNA sample. These reactions are performed in a thermocycler (M / s Perkin-Elmer 480 type). The selected thermal cycler parame...
Embodiment 3
[0151] Generate spectra of differentially expressed genes through differential display technology: identification of differentially expressed genes
[0152] The different cDNA subtypes of the dormant and non-dormant RT products obtained in Example 2 were amplified in radiolabeled dNTPs to pass polymerase chain reaction (hereinafter referred to as PCR; the PCR method is a patent owned by Hoffman-La Roche). Protect) label the amplified product. Radioactive PCR was completed in 20μl reaction mixture, which included (1) reaction buffer [10mM Tris-Cl (pH 8.4), 50mM KCl, 1.5mMMgCl 2 , 0.001% gelatin], (2) 2μM dNTP, (3) 0.2μM T 11 M and (4) 0.2μM arbitrary primers (chemical reagents 1-4 are purchased from M / s.GenHunter of Nashville, USA as part of the RNA imaging kit), 0.2μl α[ 33 P] dATP (approximately 2000 Ci / mmole, purchased from JONAKI Center, India, CCMB campus Hyderabad), and 1.0 unit of Thermophyte (hereinafter referred to as Taq) DNA polymerase (purchased from M / S. Qiagen, German...
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