CN111218454A

CN111218454A - GhRFP1 gene and recombinant vector thereof

Info

Publication number: CN111218454A
Application number: CN201811422708.0A
Authority: CN
Inventors: 王海云; 夏桂先; 李文博; 仲萌萌; 王娟; 高鹏
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2020-06-02
Anticipated expiration: 2038-11-27
Also published as: CN111218454B

Abstract

The invention discloses a GhRFP1 gene and a recombinant vector thereof, wherein the nucleotide base sequence of the gene is shown as SEQ ID NO.1 or the amino acid sequence is shown as SEQ ID NO. 2. The new gene GhRFP1 and its recombinant vector provided by the invention are introduced into cotton to increase the length of cotton fiber.

Description

GhRFP1 gene and recombinant vector thereof

Technical Field

The invention relates to the technical field of cotton gene improvement.

Background

Cotton fibers are a major source of natural textile fibers worldwide. About 70 countries worldwide grow cotton, and the annual occupied area reaches 3300 ten thousand hectares. With the improvement of living standard, people have higher and higher requirements on high-grade fiber fabrics, so that the improvement of fiber quality is one of the main targets of cotton breeding at present. Because the traditional breeding method has long period and many limitations and the strain obtained by hybridization is difficult to stabilize, the directional improvement of the fiber quality by using a genetic engineering method is a research trend.

The fiber is a single cell formed by epidermal cells of the cotton ovule exocarpium peritectum through four developmental stages of differentiation protrusion, elongation, secondary wall thickening and dehydration maturation. About 25-30% of the epidermal cells on the surface of the ovule differentiate to develop into fibroblasts, and then the ovule is elongated at a very fast speed, wherein the length/diameter can reach 1000-3000, and the final length can reach 6 cm. The secondary wall already starts to synthesize a lot before the fiber elongation stops, and the content of cellulose at maturity can reach 130ng/mm (only 1ng/mm before the secondary wall starts to synthesize). The development state of the cotton fiber in each period is closely related to the final quality of the fiber, the development of the fiber in the elongation period determines the length of the fiber, and the development of the secondary wall synthesis period determines the strength character of the fiber.

RING proteins are a class of zinc finger proteins (zinc finger proteins). The first reported RING protein was encoded by the RING1(Really interacting New Gene 1) Gene identified on the human chromosome. Such proteins have a particular cysteine-rich RING domain. The RING domain has unique characteristics different from other zinc finger proteins, and typical RING domains are C3HC4 type and C3H2C3 type, and are composed of eight conserved residues to combine two Zn ions. In plants, RING protein families are huge, and prediction is carried out according to genome sequences, so that 469 genes in Arabidopsis for coding proteins containing one or different types of RING structural domains account for 2 percent of all protein-coding genes in Arabidopsis; there are 488 such genes in rice. In addition to the RING domain, a considerable part of RING proteins also have one or several other domains with reported or unknown functions, such as some protein-protein interaction domains, ubiquitin-binding or nucleic acid-binding domains, etc. RING proteins have a wide range of actions in plants, including plant growth and development processes, plant hormone signaling pathways, plant response to biotic and abiotic stresses, and the like. At present, the function of RING proteins in cotton growth and development is poorly understood.

Disclosure of Invention

An object of the invention is to provide a GhRFP1 gene, the nucleotide base sequence of which is shown in SEQ ID NO.1 or the amino acid sequence of which is shown in SEQ ID NO. 2.

Another object of the present invention is to provide two recombinant vectors, which are obtained by inserting the above genes into an expression vector, and specifically include the following:

1) inserting nucleotides from 68 th to 667 th positions of a sequence 1 from the 5' end in a sequence table into the positions of nucleotides 8 th and 16 th positions of a pPZP-GFP vector to obtain a recombinant vector pPZP-GFP-GhRFP 1;

2) the 68 th to 667 th nucleotides from the 5' end of the sequence 1 in the sequence table are inserted between the 285 th and 277 th nucleotide sites of the pCang-HA vector to obtain the recombinant vector pCang-GhRFP 1.

The novel gene GhRFP1 provided by the invention is preferentially expressed in the elongation stage of cotton fibers; GhRFP1 was expressed at high levels in wild type fibers compared to those in the short-fiber mutants. The coding gene is introduced into cotton to increase the length of cotton fiber.

Drawings

FIG. 1 is an expression analysis diagram of GhRFP 1.

A, GhRFP1 specific expression analysis chart in different organs and tissues of cotton.

B, a graph comparing the expression levels of GhRFP1 in wild type and ligon mutant fibers.

FIG. 2 is a subcellular localization analysis chart of GhRFP 1.

Detailed Description

Example 1

First, cotton GhRFP1 gene

A cDNA encoding a RING protein family gene is separated from fibers of upland cotton R15(Gossypium hirsutum, Cotton research institute of agricultural sciences, Shanxi province) in the elongation period 12 days after flowering (12DPA), the gene is named as GhRFP1, the nucleotide-base sequence of the gene is shown in SEQ ID NO.1, the Open Reading Frame (ORF) of the gene is nucleotides 68 to 667 from the 5' end of a sequence 1 of a sequence table, the gene is named as GhRFP1, the amino acid sequence of the gene is shown in SEQ ID NO.2, and a sequence 2 in the sequence table consists of 199 amino acids.

Second, expression analysis of GhRFP1 Gene

Extracting RNA of cotton tissues and organs (roots, stems, leaves and flowers) and cotton fibers with different development days, carrying out reverse transcription to obtain cDNA, taking the 3 'end and 3' untranslated region sequences of ORF of GhRFP1 as primers (upstream primer: 5'-GGTCTTGTCCTGTTTGTCGGA-3'; downstream primer: 5'-CACCACCTCCGATCAGCATC-3'), taking cotton histone3 gene as internal reference, taking the primers of the internal reference as (upstream primer: 5'-GCCAAGCGTGTCACAATTATGC-3'; downstream primer: 5'-ACATCACATTGAACCTACCACTACC-3'), and obtaining the relative expression quantity of the GhRFP1 gene. The results are shown in FIG. 1, with the abscissa representing ovules and fibres from different organs and days of development of cotton, respectively. -3, 0, 3 are ovules 3 days before flowering, on the day of flowering and 3 days after flowering; 6, 9, 12, 15, 18, 21, 24 are fibers at different days after opening (DPA), the results are the average of three independent experiments; as can be seen, the GhRFP1 gene has the highest 12DPA expression level in the cotton fiber elongation stage. Meanwhile, the expression levels of GhRFP1 in the non-long-velvet mutant ligon and wild-type fibers thereof at different periods of fiber development are analyzed, compared with wild-type fibers, the expression level of GhRFP1 in the ligon fibers at the same period is always at a lower level, and the expression levels of GhRFP1 in ligon are respectively 36.8%, 28.1% and 31% of that in the wild-type fibers at 6, 12 and 18DPA, which indicates that the gene plays an important role in the development process of cotton fibers and can increase the length of the cotton fibers.

Example 2

Subcellular localization analysis of GhRFP 1: RNA from upland cotton R15 fiber was extracted and reverse transcribed. The cDNA was used as a template, 5'-CGGGATCCATGGCAACCCCTCCCCTTTCAT-3' as an upstream primer and 5'-CGAGCTCAGCCAAAAGTCACCACCACCTC-3' as a downstream primer ', to obtain a 612bp PCR product (sequenced to have nucleotides 68 to 667 from the 5' end of sequence 1 in the sequence listing).

Construction of pPZP-GFP-GhRFP 1: the PCR product of 612bp is cut by BamH I and Sac I, the obtained cut product is connected with pPZP-GFP carrier which is cut by the same enzyme (GhRFP1 fragment is inserted between the 8 th and 16 th bases of the carrier, the carrier is provided with complete GFP ORF nucleotide fragment), the recombinant carrier pPZP-GFP-GhRFP1 is obtained, the carrier is obtained by inserting the 68 th to 667 th nucleotides from the 5' end of the sequence 1 in the sequence table into the cut sites of BamH I and Sac I of the pPZP-GFP carrier.

The pPZP-GFP-GhRFP1 plasmid is introduced into agrobacterium EHA105 to obtain recombinant bacteria. The pPZP-GFP-GhRFP1 is used for transforming the tobacco suspension cell BY-2 BY an agrobacterium-mediated method. BY-2 cells transfected with pPZP-GFP-GhRFP1 were observed using a confocal fluorescence microscope. The excitation wavelength of GFP was 488 nm. The results are shown in FIG. 2, where GhRFP1 is located on the cell membrane.

The above examples show that GhRFP1 is a membrane protein, and has important function in the process of cotton fiber elongation growth, which results in the increase of cotton fiber length.

Examples 3,

Construction and functional identification of GhRFP1 plant expression vector

1. Construction of recombinant plant expression vectors

Construction of pCang-GhRFP 1: and (2) digesting the PCR product by using BamH I and Sac I, connecting the obtained digested product with a pCang-HA vector subjected to the same digestion to obtain a recombinant vector pPZP-GhRFP1, wherein the vector is obtained by inserting nucleotides 68 th to 667 th from the 5' end of a sequence 1 in a sequence table into the BamH I and Sac I digested sites of the pCang-HA vector.

2. Acquisition and characterization of transgenic Cotton

The recombinant plasmid pCang-GhRFP1 is introduced into agrobacterium EHA105 to obtain recombinant pCang-GhRFP1/EHA 105. Aseptic seedling lateral roots of upland cotton R15 are used as explants to be co-cultured with agrobacterium pCang-GhRFP1/EHA105, T0 cotton seedlings are obtained through callus culture and differentiation, and T0 seeds are obtained through grafting. RNA of fiber 12 days after wild type and GhRFP1 cotton blossoming is extracted, and cDNA obtained by reverse transcription is used as a template. RT-QPCR was performed using the GhRFP1 gene primer (forward primer: 5'-ATGGCAACCCCTCCCCTTTC-3'; reverse primer: 5'-TCACCACCACCTCCGATCAG-3') and Ghhisston 3 as internal reference (forward primer: 5'-GCCAAGCGTGTCACAATTATGC-3', reverse primer: 5'-ACATCACATTGAACCTACCACTACC-3') primers. The histone3 gene is used as an internal reference, and the relative expression quantity of the GhRFP1 in wild type and transgenic cotton fibers is calculated. The results show that the relative expression level of GhRFP1 in three lines of T1 generation cotton is far higher than that of the wild type, which indicates that the cotton which over-expresses GhRFP1 is obtained.

3. Phenotypic analysis of GhRFP 1-transgenic cotton

Seed cotton in naturally mature wild type and transgenic cotton bolls is selected for phenotype observation. The mature fibers were combed out and compared in length, and the GhRFP 1-converted cotton fibers were found to be slightly longer than the wild type. The detection of a cotton fiber performance tester shows that compared with the wild cotton fiber, the quality (length and strength) of the transgenic cotton fiber is increased to different degrees, wherein the length is increased by 5-7%.

Line of plants	Wild type	Transgenic line 1	Transgenic line 2	Transgenic line 3
					Length (mm)	28.43	30.43	29.83	30.09
Strength (cN/tex)	29.19	31.48	30.67	31.41

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> GhRFP1 gene and recombinant vector thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>810

<212>DNA

<213> upland cotton (Gossypium hirsutum)

<400>1

acgcggggct ttttgttttc tgtgtttttt tttttagtcc ccaaatggat tctccctttc 60

cgttcaaatg gcaacccctc ccctttcatc ccctacttat ctcaccacca atttaggcgt 120

cggctacgcc atcgtcgtcg ccctcggctt cctcctcctc ctctccactc tcctcctcgc 180

ttcctacatt tgcttccgct cttcctcacc cctttcacct cgtgccattt ccccaaaccc 240

catttcaaca cccaccgccg ccaactccga cggcatcgtc ctccccagga ttgttttcat 300

cggcgaagaa gaccatgaaa acgacgagga aaacgtcgtc gtctttgggc ttgatcgtgc 360

cgtcataaac tcttacccca agttccggtt cagcaaaaag gaggtgtcta cggcggtgga 420

ggtgacgggt tctagtgctg ggaacaccat ttgttcgata tgtttgtgtg agtatagaga 480

atcagagatg ttgaggatga tgcctgaatg taggcattgt tttcatgtaa cttgtattga 540

tgcttggctg aaacttaatg ggtcttgtcc tgtttgtcgg aactcaccgc ttccgacacc 600

gatttctacg cctttgtcgg aagttgtacc tttgtctcag tacgatgctg atcggaggtg 660

gtggtgactt ttggcttttg ggttaccatc tttttcattt ttttttcttc aatttttttc 720

ctgtgtggaa atttggtttt ttgtcatgga tgcttttttt tggctcaatg aatggtgatt 780

tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa 810

<210>2

<211>198

<212>PRT

<213> upland cotton (Gossypium hirsutum)

<400>2

Met Ala Thr Pro Pro Leu Ser Ser Pro Thr Tyr Leu Thr Thr Asn Leu

1 5 10 15

Gly Val Gly Tyr Ala Ile Val Val Ala Leu Gly Phe Leu Leu Leu Ser

20 25 30

Thr Leu Leu Leu Ala Ser Tyr Ile Cys Phe Arg Ser Ser Ser Pro Leu

35 40 45

Pro Pro Arg Ala Ile Ser Pro Asn Pro Ile Ser Thr Pro Thr Ala Ala

50 55 60

Asn Ser Asp Gly Ile Val Leu Pro Arg Ile Val Phe Ile Gly Glu Glu

65 70 75 80

Asp His Glu Asn Asp Glu Glu Asn Val Val Val Phe Gly Leu Asp Arg

85 90 95

Ala Val Ile Asn Ser Tyr Pro Lys Phe Arg Phe Ser Lys Lys Glu Val

100 105 110

Ser Thr Ala Val Glu Val Thr Gly Ser Ser Ala Gly Asn Thr Ile Cys

115 120 125

Ser Ile Cys Leu Cys Glu Tyr Arg Glu Ser Glu Met Leu Arg Met Met

130 135 140

Pro Glu Cys Arg His Cys Phe His Val Thr Cys Ile Asp Ala Trp Leu

145 150 155 160

Lys Leu Asn Gly Ser Cys Pro Val Cys Arg Asn Ser Pro Leu Pro Thr

165 170 175

Pro Ile Ser Thr Pro Leu Ser Glu Val Val Pro Leu Ser Gln Tyr Asp

180 185 190

Ala Asp Arg Arg Trp Trp

195

Claims

1. The GhRFP1 gene has the nucleotide base sequence as shown in SEQ ID No.1 or the amino acid sequence as shown in SEQ ID No. 2.

2. A recombinant vector pPZP-GFP-GhRFP1 is obtained by inserting nucleotides 68 to 667 from the 5' end of a sequence 1 in SEQ ID NO.1 between the 8 th and 16 th nucleotide sites of a pPZP-GFP vector to obtain a recombinant vector pPZP-GFP-GhRFP 1.

3. A recombinant vector pCang-GhRFP1 is obtained by inserting the 68 th to 667 th nucleotides from the 5' end of the sequence 1 in SEQ ID NO.1 between the 285 th and 277 th nucleotide sites of the pCang-HA vector to obtain the recombinant vector pCang-GhRFP 1.