CN114990119A

CN114990119A - Long-chain non-coding RNA gene of rape and application thereof in increasing grain weight of rape seeds

Info

Publication number: CN114990119A
Application number: CN202210737422.1A
Authority: CN
Inventors: 鲁少平; 郭亮; 李雨晴
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-06-27
Filing date: 2022-06-27
Publication date: 2022-09-02

Abstract

The invention discloses a long-chain non-coding RNA gene MSTRG.22563 of rape, the nucleotide sequence of which is shown as SEQ ID NO. 1. By obtaining an over-expression material and analyzing the thousand grain weight of the transgenic material, the MSTRG.22563 gene is found to improve the grain weight of the brassica napus seeds. The invention further provides a method for increasing the grain weight of rape seeds, which comprises the following steps: the overexpression vector containing the MSTRG.22563 gene is transformed into the genome of the crop by using an agrobacterium-mediated genetic transformation method to obtain the rape variety with the MSTRG.22563 gene overexpressed. The invention has potential application value in rape high-yield breeding.

Description

Long-chain non-coding RNA gene of rape and application thereof in increasing grain weight of rape seeds

Technical Field

The invention belongs to the field of molecular breeding, and particularly relates to a long-chain non-coding RNA gene MSTRG.22563 of rape and application of the gene in regulation and control of grain weight of rape seeds.

Background

Long non-coding RNAs (lncRNAs) were originally thought of as transcription waste without any function, and the role of lncRNAs in organisms has been more concerned since the end of the 20 th century 80 years when H19 was found to have RNA function in mammals. Over the past decades, an increasing number of lncRNAs have been found to play a key role in maintaining the normal function of animal cells. Recently, large numbers of lncRNAs have also been found in many plants, as 6480, 2224, 14749, 50873, 20163 and 3181 lncRNAs were predicted in arabidopsis, rice, cotton, peanut, maize and canola, respectively. The functional characteristics of some lncRNAs in plants are related to the growth, development and response to abiotic and biotic stress of the plants.

Rapeseed is the third largest oil crop in the world, accounting for about 13% of the world's oil production. The three factors that contribute to rape yield are the number of siliques per unit area, the number of grains per silique and the grain weight. Therefore, the increase of the grain weight of the rape directly influences the yield of the rape. Grain weight is a quantitative trait controlled by multiple genes, and is regulated and controlled by environment and genotype.

The invention clones the long-chain non-coding RNA gene MSTRG.22563 from rape, and researches show that the overexpression of the MSTRG.22563 gene in rape can obviously improve the seed weight of rape and provide theoretical basis for high-yield breeding of rape.

Disclosure of Invention

The invention aims at providing a rape long-chain non-coding RNA gene which is named as MSTRG.22563 gene by the applicant, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the sequence length is 246 bp. The gene is finally obtained by screening and identifying a large number of rape lncRNAs, and can be obtained by amplifying rape genome, mRNA and cDNA by adopting a PCR technology.

The invention further provides a method for cloning the gene, which comprises the following steps:

(1) extracting total RNA from rape seeds;

(2) reverse transcription into cDNA;

(3) designing positive and negative primers to amplify target gene from cDNA.

Wherein, the forward primer: 5'-GCGACTAGT CGTCGAGCTCGGTAGCGTG-3' (SEQ ID NO:2)

Reverse primer: 5'-GCGTTCGAA CCCCGTTATCCTTCCACCGT-3' (SEQ ID NO:3)

The invention also provides a recombinant plasmid and a recombinant bacterium containing the rape long-chain non-coding RNA gene.

The rape long-chain non-coding RNA gene, the recombinant plasmid or the recombinant strain provided by the invention can be used for carrying out molecular breeding on rape and improving the grain weight of rape seeds.

The invention further provides a method for increasing the grain weight of rape seeds, which comprises the following steps: the over-expression recombinant plasmid containing the rape long-chain non-coding RNA gene is transformed into a rape genome by using an agrobacterium-mediated genetic transformation method to obtain a rape variety with the rape long-chain non-coding RNA gene over-expression, wherein the nucleotide sequence of the rape long-chain non-coding RNA gene is shown as SEQ ID NO. 1.

The vector plasmid used in the present invention refers to any vector known in the art capable of expression in plants, for example, suitable vectors for constructing the over-expression recombinant plasmid of the present invention include, but are not limited to, PMDC83 and the like.

One characteristic of the invention is that the thousand seed weight of the transformed material is analyzed by obtaining the over-expression material, and MSTRG.22563 is found to be regulating the thousand seed weight of rape seeds.

The high grain weight overexpression rape plant obtained by the invention has potential application value in rape high-yield breeding.

Drawings

FIG. 1: agarose gel electrophoresis detection result of MSTRG.22563 gene amplification product.

FIG. 2: the plasmid map of the constructed plant expression vector pMDC 83-MSTRG.22563.

FIG. 3: and (3) qRT-PCR detection of the rape transformed single plant. OE4, OE5, OE6, OE7 and OE8 are overexpression lines of the gene mstrg.22563. Each strain had 3 different individual strains, representing P <0.01 in Student's t test, representing P < 0.05.

FIG. 4: MSTRG.22563 was characterized in the phenotype of oilseed rape. Analyzing the thousand seed weight of rape seeds by using a thousand seed weight analyzer, wherein OE4, OE5 and OE7 are overexpression strains of the gene MSTRG.22563. There were 8-12 different individuals per strain, representing P <0.05 in Student's t test.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as the Instrument book molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory,1989) or the procedures suggested in the manufacturer's manual were followed.

Example 1 cloning of MSTRG.22563 Gene

(1) Extraction of RNA

Total RNA was extracted using TransZol (catalog number ET101) from general gold, according to the protocol of kit instructions. The reagents to be prepared in advance were RNase-free water, chloroform, isopropanol and 75% ethanol (prepared from DEPC-treated water), and the reagents and laboratory articles were all treated with DEPC-inactivated RNase. The method comprises the following specific steps:

a. fully grinding developing seeds of the brassica napus in liquid nitrogen until the seeds are powdery, transferring 100mg of the ground sample into a 1.5ml centrifuge tube, adding 1ml of TransZol, then violently shaking up and down to fully mix the seeds uniformly, homogenizing, and then standing for 5 minutes at room temperature;

b. adding 0.2ml of chloroform into the centrifuge tube, violently shaking for 15 seconds, and incubating for 3 minutes at room temperature;

c.10, 000 Xg at 4 ℃ for 15 minutes;

d. transferring the upper colorless aqueous phase into a new centrifuge tube (with the volume of about 0.6ml), adding 0.5ml of precooled isopropanol, reversing, mixing uniformly, and incubating for 10 minutes at room temperature;

e.10, centrifuging at 4 ℃ at 000 Xg for 10 minutes, pouring out the supernatant, and forming precipitates on the side wall and the bottom of a centrifugal tube; add 1ml of 75% ethanol (DEPC water) to the centrifuge tube and vortex vigorously;

f.7, centrifuging at 500 Xg for 5 minutes at 4 ℃, removing supernatant, and airing and precipitating at room temperature for about 15 minutes;

g. dissolving the precipitate in 50-100 μ l RNA dissolving solution, incubating at 55-60 deg.C for 10min, and storing in-80 deg.C refrigerator.

h. Taking 1 μ l of extracted total RNA to determine RNA concentration and quality under Nanodrop, and identifying RNA purity according to 1.8< OD260/OD280< 2.0. At the same time, 1. mu.l of the suspension was subjected to 1% agarose gel electrophoresis to examine its integrity and quality.

(2) Reverse transcription of RNA

The reverse transcription kit used in the experiment is full-scale gold

One-Step gDNAremoval and cDNASynthesis SuperMix (catalog No. AE 311-03). The specific experimental procedures followed the instructions:

mu.l of adsorbed Oligo (dT)18Primer, 10. mu.l of 2 × ES Reaction Mix, and 1. mu.l of the same were added in this order using 5. mu.g of total RNA as a template

RT/RI Enzyme Mix, 1. mu.l of gDNAremover, finally supplemented to 20. mu.l with RNase-free Water. Gently mixing the reaction systems uniformly, placing at 42 ℃ and incubating for 30min, and synthesizing first chain cDNA and removing gDNA; then, the mixture was heated at 85 ℃ for 5 seconds to deactivate

RT/RI and gDNARemover. Finally, 180. mu.l of RNase-free Water was added to dissolve the synthesized cDNA.

(3) Amplification of MSTRG.22563 Gene

Using the cDNA as template, forward primer 5'-GCGACTAGTCGTCGAGCTCGGTAGCGTG-3' and reverse primer 5'-GCGTTCGAACCCCGTTATCCTTCCACCGT-3' were used to amplify a full-length fragment containing MSTRG.22563. By using I-5 ^TM 2 × High-Fidelity Master Mix (TSINGKE Biologica technology) for PCR amplificationAnd (5) increasing.

The PCR amplification reaction system is as follows:

preparing a reaction solution in a PCR tube according to the reaction system, and carrying out amplification on a Bio-Rad PCR instrument, wherein the PCR amplification program comprises the following steps:

the amplified product is detected by 1% agarose gel electrophoresis (figure 1), the MSTRG.22563 full length of 246bp is obtained by amplification, and the heaven root agarose gel DNA recovery kit is used for gel digging and recovery of the PCR amplified product.

Example 2 construction of transformation vector for overexpression of MSTRG.22563 Gene

(1) The MSTRG.22563 fragment obtained above was digested simultaneously with the fast restriction enzymes SpeI and BstbI, and the restriction enzyme used in this experiment was a product of Shanghai Saimer Feishell science and technology (China). The double enzyme digestion system is as follows:

the cleavage reaction was carried out in a 37 ℃ water bath for 1.5 hours. The enzyme digestion product was recovered by using a general-purpose DNA purification recovery kit (DP130227) from Beijing Tiangen bioengineering Co.

(2) The enzyme digestion product is connected to the vector plasmid PMDC83, and the connection reaction system is as follows:

the connection reaction conditions are as follows: 3 hours at 22 ℃.

(3) Transforming Escherichia coli DH5 alpha, screening positive clones, performing quality-improving cutting identification, selecting 3 positive clones, sending samples, sequencing, and analyzing to show that the sequence of MSTRG.22563 gene is successfully connected with vector, thus successfully constructing recombinant plasmid pMDC83-MSTRG.22563 for transforming plant, wherein the plasmid information is shown in figure 2.

(4) Introducing the correctly constructed recombinant plasmid vector into agrobacterium strain GV3101, and selecting positive monoclone for preservation in a refrigerator at-80 deg.c. Agrobacterium GV3101 competence was transformed using an electric shock method, the specific experimental steps were as follows:

a. respectively cleaning the electric revolving cups with pure water, ultrapure water and absolute ethyl alcohol, and placing the electric revolving cups on a super clean bench for airing;

b. thawing the recombinant plasmid and agrobacterium GV3101 competence on ice;

c. adding 1 mul of correctly constructed recombinant plasmid into 25 mul of agrobacterium GV3101 competence, and slightly sucking, beating and uniformly mixing to avoid generating bubbles;

d. quickly transferring the mixed solution into a pre-cooled electric rotary cup along the cup wall;

e. adjusting the electric rotating instrument to 1800V, then wiping the outer wall of the electric rotating cup by using absorbent paper, and electrically shocking under the condition of 1800V voltage;

f. after the electric shock is successful, adding 200 mu l of the non-resistant LB liquid culture medium into the electric rotating cup, gently sucking for several times, then transferring the solution into a 1.5ml sterile centrifuge tube, and activating for about 2 hours at the temperature of 28 ℃ at 150 r/min;

g. coating the recovered strain on an LB solid culture medium containing antibiotics, and performing inverted culture in an incubator at 28 ℃ for 48 hours;

h. single colonies were picked on the plate, PCR amplified with the pre-vector primer and the post-target gene primer, and positive clones were detected by 1% agarose gel electrophoresis.

i. Adding the positive bacterial liquid into a resistant LB liquid culture medium, carrying out amplification culture in a shaking table at the temperature of 28 ℃ and at the speed of 150r/min until the OD value reaches 0.8, adding equal volume of 50% (v/v) glycerol, mixing uniformly, and storing in a refrigerator at the temperature of-80 ℃.

Example 3 genetic transformation experiments

(1) Genetic transformation of oilseed rape

The constructed overexpression vector is subjected to genetic transformation of rape by adopting hypocotyl dark light culture, a receptor for rape transformation in the invention is cabbage type rape Westar, and the specific method is as follows:

a. and (3) sterilizing the seeds:

pouring the selected mature and plump cabbage type rape 'Westar' seeds into a culture box, and soaking the seeds in 75% alcohol for about 1min for no more than 5 min; washing the soaked seeds with sterile water for 1-2 times; adding 0.15-0.2% mercuric chloride solution (preserved in laboratory brown bottle, and diluted with laboratory 5% mother liquor) capable of submerging seeds, and sterilizing for 15 min; washing the seeds with sterile water for 5-6 times.

b. Sowing:

sowing seeds in an M0 culture medium by using sterilized tweezers, and uniformly sowing 36-40 seeds in each culture dish; after seeding, the culture dish is placed under the dark light condition for about 7 days at the temperature of 25 ℃.

c. And (3) culturing agrobacterium:

inoculating bacteria on the resistant plate five days after sowing, dipping agrobacterium liquid containing target genes by using a sterilized inoculating loop, and scribing on a resistant LB plate; after 2 days, sucking positive single colonies on the resistant plate by using a toothpick or a gun head, placing the positive single colonies in a resistant LB liquid culture medium for blow beating, and shaking the bacteria at the temperature of 28 ℃ and at the speed of 150 r/min.

d. Preparation and infection of explants:

detecting the concentration of the bacterial liquid under the condition of light absorption at 600nm (spectrophotometer), measuring the OD value of the bacterial liquid, wherein the OD value is optimal between 0.6 and 0.8, centrifuging the bacterial liquid at 6000rpm for 10min, and discarding the supernatant; resuspending with DM (dilution Medium) liquid equal in volume to the bacteria liquid, centrifuging at 6000rpm for 10min, and discarding the supernatant; the suspension was suspended with the same volume of DM solution as the inoculum solution. Then 2ml of the bacterial liquid is taken out and put into a sterilized culture dish, and 20ml of DM solution is used for diluting the bacterial liquid (1: 10); cutting off hypocotyl of seedling after culturing in dark light for 7 days with sterile scalpel and tweezers, cutting to length of 0.8-1.0cm, cutting off explant once as much as possible, and keeping the cut neat; placing the cut explant into the prepared bacterial liquid with the concentration, infecting for 30min (the time cannot be too long), and sucking and beating once at intervals by using a liquid moving machine to ensure that the cut is fully contacted with the bacterial liquid.

e. Culturing callus tissues:

absorbing bacteria liquid on explants by using sterilized absorbent paper, transferring the explants to M1 culture medium, culturing 50-60 explants per dish under the condition of dark light for about 2 days at the temperature of 25 ℃; two days later, the explants were transferred to M2 medium inducing callus, cultured under light conditions (16 h/8 h at day/night) at 22 ℃, and then cultured under light conditions; after three weeks, transferring the explants which grow normally and have two expanded ends into a differentiation medium M3, and subculturing once every 2-3 weeks until green buds grow; cutting off green buds with obvious base nodes, transferring the green buds into a culture medium M4 for rooting, taking 2-4 weeks, transplanting the green buds into an outdoor field after rooting, and observing the growth phenotype of the plants in the field. The medium formulation used is shown in table 1.

TABLE 1 formulation of Medium for dark light culture of hypocotyls

(2) Identification of overexpressing transformants

Extracting genome DNA of the obtained rape overexpression transformation individual, detecting the insertion of the exogenous gene segment by PCR, wherein the overexpression framework vector is pMDC83, a primer PMDC83-R (5'-CCAGCAGCTGTTACAAACTCAAG-3') is designed on the framework vector, PCR (MSTRG.22563-F and pMDC83-R) is carried out by matching the vector framework primer with the exogenous segment primer, the exogenous segment primer MSTRG.22563-F (5'-GCGACTAGTCGTCGAGCTCGGTAGCGTG-3') and MSTRG.22563-R (5'-GCGTTCGAACCCCGTTATCCTTCCACCGT-3') are used for detecting the transgenic seedling at the PCR level.

The PCR reaction system is as follows:

the PCR reaction program is:

and (3) carrying out qRT-PCR on the rape transgenic positive seedlings obtained by the PCR to detect the gene expression quantity. RNA of transformed individual seeds was extracted using the TransZol kit, and reverse transcription was performed to synthesize cDNA (as in example 1), and finally PerfectStart from gold Oldham was used ^TM Green qPCR Supermix reagent was used to perform fluorescent quantitative PCR analysis on the samples.

The quantitative primers are designed by using Primer 5 software, the size of the product is between 100bp and 200bp, and BLAST comparison is carried out by using a reference sequence after the quantitative primers are designed, so that the specificity of primers MSTRG.22563qPCR-F (5'-CGTTCCAGGTTAGGAGTTGAG-3') and MSTRG.22563qPCR-R (5'-GGTGTACGCTGCTCTGTAAA-3') is ensured. BnaACTIN-F (5'-TGTTCCCTGGAATTGCTGACCGTA-3') and BnaACTIN-R (5'-TGCGACCACCTTGATCTTCATGCT-3') were used as internal reference primers for rape qRT-PCR. The qRT-PCR reaction system is as follows:

the qRT-PCR reaction program was:

the qRT-PCR reaction was performed in the Bio-Rad CFX96 Real-Time System.

The quantification of the variation between the different replicates was calculated by means of delta-threshold cyclic quantification (2-. DELTA.CT) based on normalization of the internal reference primers. The expression level of the transformed plants was analyzed as shown in FIG. 3.

(3) Thousand kernel weight analysis of transgenic plants obtained by genetic transformation

And (3) selecting more than 500 full rape seeds by using a thousand seed weighing instrument for counting and weighing.

Thousand kernel weight results analysis showed that the thousand kernel weight of the over-expressed material of mstrg.22563 gene was significantly increased by 0.18-0.47g for OE material (fig. 4) compared to WT (3.35 ± 0.31) for OE4(3.82 ± 0.21), OE5(3.53 ± 0.20) and OE7(3.66 ± 0.44), respectively. These results indicate that the MSTRG.22563 gene plays an important role in regulating thousand kernel weight of rape.

Sequence listing

<110> university of agriculture in Huazhong

<120> long-chain non-coding RNA gene of rape and application thereof in increasing grain weight of rape seeds

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> DNA

<213> Brassica napus (Brassica napus L.)

<400> 1

cgtcgagctc ggtagcgtgg gcttataatc catgtggaga caataggatg atgggacgtt 60

gggtcgatct cattgggccg ggtttgttgg gcttattatg gcctgcccgt tccaggttag 120

gagttgaggc atgcggcttg agcgaaggct ttagtcacgt gtttcttaaa gtgggggaga 180

ctgcgtgagg ctggttttac agagcagcgt acacctcccg ctcttgacgg tggaaggata 240

acgggg 246

<210> 2

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gcgactagtc gtcgagctcg gtagcgtg 28

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gcgttcgaac cccgttatcc ttccaccgt 29

Claims

1. A long-chain non-coding RNA gene of rape has the nucleotide sequence shown in SEQ ID NO. 1.

2. The primer for amplifying the long non-coding RNA gene of the rape of claim 1 has the nucleotide sequence shown as SEQ ID NO. 2 and SEQ ID NO. 3.

3. A recombinant plasmid containing the long non-coding RNA gene of Brassica napus as claimed in claim 1.

4. A recombinant bacterium comprising the long non-coding RNA gene for Brassica napus according to claim 1.

5. The rape long-chain non-coding RNA gene of claim 1, the recombinant plasmid of claim 3 and the recombinant bacterium of claim 4 are applied to increasing the grain weight of rape seeds.

6. A method for increasing the grain weight of rape seeds is characterized in that: the over-expression recombinant plasmid containing the rape long-chain non-coding RNA gene is transformed into a rape genome by using an agrobacterium-mediated genetic transformation method to obtain a rape variety with the rape long-chain non-coding RNA gene over-expression, wherein the nucleotide sequence of the rape long-chain non-coding RNA gene is shown as SEQ ID NO. 1.

7. The method for increasing the grain weight of canola as claimed in claim 6, wherein: the vector plasmid for the overexpression of the recombinant plasmid is the PMDC83 plasmid.