LmPAL1: Cloning and expression analysis of the lmPAL1 gene of the gray felt winter.

The cloning and expression analysis of the lmPAL1 gene of the gray felt-fed winter Lonicera macranthoids Hand.-Mazz.. comes from the stoic genus Lonnicera Linn.semi-evergreen entangled vine plant for the steamy winter stoice, which is attributed to the mountain silver flowers, with the effect of clear ingestion, evacuation of wind heat.
chlorogenic acid (chlorogenic acid, CGA) is the main active ingredient of gray felt felt winter, and the content is high, modern research shows that chlorogenic acid with antioxidant, antihypertensive, antibacterial, anti-tumor, anti-radiation, blood sugar reduction, blood lipids, anti-inflammatory, kidney, liver protection and other pharmacological effects, medicine, chemical and food fields have a broad demand for green raw acid.
chlorogenic acid is a natural product of the three-bit hydroxyl synthesis ester of coffee acid, and is one of the important plant phenylpropin secondary metabolites, initially produced by L-phenylalanine in phenylalanine (phenylalanine-ammonia-lyase, PAL) to produce a reaction cinnamon acid, then cinnamon-4 hydrosoziase (c. e, C4H) and 4-coumarate coenzyme (4-coumarate CoA lili, 4CL) to produce a pair-coenzyme A, and finally hydroxycoquine coenzyme a quinycosic cinnamon transferase (hydroxyinnamoyl-CoAquiateycoysycycoinnainnalithia, 4Cl) to produce a green acid .
there are currently researchers who have studied PAL gene cloning, bioinformatics and expression pattern analysis in plants such as the stoic winter, Du Zhong, Dusan, Dion, Black Triangle, Fine Sin, Turmeric, and Danghui, etc.
consult the literature and NCBI database found that the NCBI has uploaded gray felt winter PAL1 part of the core sequence, only 493 bases, coding 164 amino acids, currently no gray felt to endure winter PAL1 gene full-length cloning reports.
this study to gray felt stoic winter flower as a material, through the early gray felt felt winter transcription group data Unigene sequence design primer, using RT-PCR and RACE technology, clone gray felt winter PAL1 (LmPAL1) gene full-length cDNA sequence, and bioinformatics analysis, the use of real-time Fluorescent quantitative PCR (qRT-PCR) analyzes the expression pattern of different flowering periods and different organs of gray felt felt winter, which provides the basis for further improving the content of gray felt winter chlorogenic acid content and the preferred seed resources through genetic engineering, and also provides experimental data for the study of the biosynthesis method and adjustment mechanism of chlorogenic acid.
1, materials and reagent gray felt winter seed in Hunan University of Traditional Chinese Medicine pharmaceutical plant garden, in late May 2018 to harvest the year's fresh stems, leaves and 7 different flowering periods (early buds, turquoise flowering, green and white buds, white buds, white flowering period, golden flowering period Period, withering period) flower samples, with liquid nitrogen filled with foam boxes shipped back to the laboratory, by Hunan University of Traditional Chinese Medicine, Professor Sunday Bao identified as gray felt winter Lonicera macraneoids Hand.-Mazz., preserved in the ultra-low temperature refrigerator of 80 degrees C.
Polysaccharide Plant Total RNA Extraction Kit (Hangzhou Bozhi Technology Co., Ltd.); RevertAid First Strand cDNAKit Reverse Record Kit (Thermo Company); Rapid Agar Sugar Gel DNA Recovery Kit, 2 x Taq Master Mix (Dye), 2 x Pfu MasterMix (Dye) Kang for Century Biotech) ;p EASY® -T1 Cloning Vector, pEASY® - Blunt Cloning Vector, TranStart® Green QPCR SuperMix UDG (Beijing All-Golden Biotech Co.®, Ltd.); The
primer sequence is shown in Table 1, synthesized by Shanghai Bioengineering Co., Ltd., with a concentration of 10 ?mol/L.
2, method 2.1 LmPAL1 gene cloning 2.1.1 total RNA extraction and cDNA synthesis to take the appropriate amount of gray felt to endure winter buds, according to polysaccharide plant total RNA extraction kit specification to extract total RNA, with 1.5% agar gel electrophoresis to detect RNA integrity, nucleic acid analyzer to detect RNA purity.
will be better quality RNA, refer to the First Chain of Reverse Synthetic CDNA by reference to the Buk Aid FirstAid Strand cDNA KitSynthesis specification.
2.1.2 LmPAL1 gene core fragment amplification Based on the pre-transcription group sequencing results of this group, selected the sequence noted as phenylalanine deaminase, and used Primer Premier 5.0 to design specific prime prime prime primee PAL1-F and PAL1-R (Table 1) to reverse the resulting cDNA as a template for PCR amplification.
PCR reaction system (25?L) :d dH2O9.5,l, 2 x Taq MasterMix (Dye) 12.5?L, cDNA 1?L, PAL1-F and PAL1-R each at 1?L.
PCR reaction conditions: 94 degrees C pre-degeneration 2 min; 94 degrees C degeneration 30 s, 60 degrees C annealing 30 s, 72 degrees C extension 1 min, cycle 35 times; 72 degrees C final extension 10 min, 4 degrees C preservation.
PCR products are tested with agarose gel electrophoresis, cut off the purpose strip, and quick agar gel DNA recovery kit is recycled, with pEASY®-T1Clon Vectoring as the carrier, connected to the target gene, converted into the receptor cells Through the LB solid media containing X-gal, IPTG, Amp for blue-white spot screening, selection of white spots for colony PCR, positive cloning inoculated in the LB liquid medium containing Amp overnight culture, commissioned Platinum Biotechnology Co., Ltd. sequencing.
2.1.3 LmPAL1 Gene 3' and 5' end RACE amplification based on the "2.1.2" core fragment sequencing results, design race-specific primer PAL1-3' and PAL1-5' (Table 1), first according to Clontech company SMARTERTERRACE®race5'/3'Kit'Kit kit kit instructions, obtained LmPAL1 gene 5' and 3'-Ready-RACE-sand-
PCR reaction system (50?L) :P CR-Grade H2O15.5,2 x SeqAmp Buffer 25?L, SeqAmp DNA Polymerase 1.0?L, 5' or 3'-RACE Ready-cDNA 2.5?L, 10 x UPM 5'L, 5'or 3'
PCR reaction conditions: 94 degrees C, 30 s, 72 degrees C 2 min, 5 cycles;
the process of band recovery and sequencing of the amplification purpose is the same as the "2.1.2" item, where the carrier uses pEASY®-Blunt VectorCloning.
2.1.4 LmPAL1 gene cDNA full-length sequence verification using Conting Express for sequence stitching of 3'end and 5' ends, obtaining the cDNA length of the LmPAL1 gene, and designing cDNA full length verification primers V-PAL1-F and V-PAL1-R (Table 1) according to the splicing full length design, with cDNA as the template under "2.1.1".
Reaction System (25?L) :d dH2O 9.5?L, 2 x Pfu MasterMix (Dye) 12.5?L, cDNA 1?L, V-PAL1-F, and V-PAL1-R each 1?L.
PCR reaction conditions: 94 degrees C pre-degeneration 2 min; 94 degrees C degeneration 30 s, 60 oC annealing 30 s, 72 degrees C extension 3 min, cycle 35 times; 72 degrees C final extension 5 min, 4 degrees C preservation.
the process of band recovery and sequencing of the amplification purpose is the same as the "2.1.2" item, where the carrier uses pEASY®-Blunt VectorCloning.
2.2 Bioinformatics Analysis uses NCBI online software "ORFfinder" () to find The Open Reading Box of HQT, predict protein structure through online software ProtParam() and analyze the amino acid composition, protein relative molecular mass, theoretical and other electrical points and stability parameters of the target gene-coded protein; TMHMM 2.0() protein transmembrane structure analysis, SignalP 4.1 Server() predictive signal peptides, InterProScan() analysis of protein domains, SOPMA() and SWISS-MODEL software predict the secondary structure and tertiary structure of proteins, blast through NCBI protein sequence database, screening of highly homogenous species, using MANDNA software for amino acid multi-sequence comparison. , MEGA 7 software builds the system evolution tree.
2.3 The tissue expression level analysis of the LmPAL1 gene is based on the full length sequence of the LmPAL1 gene, and the specific primers Q-PAL1-R and Q-PAL1-F (Table 1) of qRT-PCR are designed, with 18 S rRNA as the endofor gene.
the total RNA of gray felt-fed winter stems, leaves, and 7 different flowers, according to the "2.1.1" method, and the RNA was quantified, reverse-recorded into cDNA for real-time fluorescence quantitative analysis.
Reaction System (20?L): TranStart® Green qPCR SuperMix UDG 10?L, cDNA 1?L, Q-PAL1-R and Q-PAL1-F each 0.4?L, ddH2O8.2.L.
reaction conditions: 50 degrees C, 2 min, 94 degrees C, 10 min; 94 degrees C, 5 s, 55 degrees C, 15 s, 72 degrees C, 10 s, 40 cycles, meltcurve analysis, each sample repeated 3 times, the experiment in Bio-Rad CFX96, using 2 - ct to calculate the relative expression of the gene.
3, results and analysis of 3.1 LmPAL1 gene cloning 3.1.1 total RNA extraction ash felt winter flower total RNA agar sugar gel electrophoresis See 1, visible 28 S and 18 S strip slicing obvious, of which 28 S strip The band brightness is 2 times that of 18 S, indicating better RNA integrity, with A260/A280 values of 1.8 to 2.0, A260/A230 values greater than 2.0, RNA quality in line with subsequent experiments.
3.1.2 LmPAL1 core fragment amplification through transcription group Unigene sequence design primers, using RT-PCR amplification to obtain a single band, about 1100bp (Figure 2), after recycling, purification, cloning, sequencing, obtained the fragment sequence of 1,163 bp, through DNA MAN comparison, consistent with transcription group sequencing results, similarity of up to 100%.
3.1.3 The acquisition of the whole length cDNA of the LmPAL1 gene is designed with RACE-specific primers according to the core fragment sequence, with 5'end and 3' end amplification, respectively.
3' end appears around 1,463 bp with a bright band, and 5' end appears around 1,167 bp.
will be the bright belt cut glue recovery, converted to the carrier, select editing positive clone sequencing, 3'end, 5' end and core fragment sequence stitched together, to obtain the LmPAL1 gene cDNA full-length sequence 2 549 bp (Figure 3).
design full-length verification primer, pcR amplification with the cDNA under "2.1.1" as a template, with a single bright band with a clear light band of about 2,500 bp, the bright band recovery, purification, cloning, sequencing, sequencing results and stitching full-length sequence consistent, indicating the successful cloning of the Lm1 gene PALcDNA full length.
3.2 LmPAL1 Gene Bioinformatics Analysis 3.2.1 LmPAL1 Protein Physical and Chemical Properties LmPAL1 Gene Length 2,549bp, 3'end non-coding region 202bp, with 30 bp ployA tail, 5' end non-coding region 202 bp, with a complete open reading box 2 145 bp, the sequence encoded amino acid 714, alkali and amino acid sepsis.
uploaded it to the NCBI database and named it LmPAL1, with GenBank login number MH236488.
used ExPASy ProtParam online software to predict the physical and chemical properties of the LmPAL1 gene-coded protein, and speculated that its molecular formula was C3420H5482N950O1043S29, the relative molecular mass was 77 526.63, the isoelectric point was 6.04, and predicted that it was in mammals. Half-life is 30 h, half-life in yeast is greater than 20 h, instability coefficient is 35.26 to 40, is a stable protein, with positive charge residuals (Arg-Lys) is 73, negative-charged residuals (Asp-Glu) is 83, fat coefficient 91.25, hydrophilic average system.