CN111675757A - Du pear vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement - Google Patents

Abstract

The invention provides a birch-leaf pear vacuole proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement, belonging to the technical field of genetic engineering. The amino acid sequence of the birchleaf pear vacuole proton pump PbVHA-B1 is SEQ ID No.1, and the nucleotide sequence of the coding gene is SEQ ID No. 2. PbVHA-B1 or coding gene thereof in salt-tolerant plant breeding or breeding transgeneThe application in salt-tolerant plants. The relative expression quantity of the coding gene is gradually increased along with the prolonging of the NaCl treatment time in 12 hours by adopting a real-time quantitative PCR method for analysis, which shows that the coding gene has the salt resistance function in response to the salt stress treatment. The overexpression of the coding gene can effectively maintain intracellular Na⁺/K⁺The concentration balance keeps the cell osmotic potential stable, so that the plant can better adapt to salt stress, and the silencing of the coding gene reduces the active oxygen scavenging capacity of the plant, so that the salt tolerance of the plant is weakened.

Description

Du pear vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a birch-leaf pear vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement.

Background

Pears are one of the fruit trees mainly planted in the world, and are the third largest fruit next to apples and oranges in China, the planting area is particularly wide, and a three-area and four-point production area layout mode that the pears are planted from the northeast to the Guangxi and from the Yunnan to the Shandong is formed. Although the layout and the planning of the pear dominant production area can well promote the development of the pear industry in China, the development of the pear industry is influenced by salt, alkali, drought, freezing damage and flooding due to the difference of environmental factors in different areas. Therefore, whether to culture a new variety with excellent stress resistance is crucial to the benign sustainable development of pear industry. The conventional breeding method cannot meet the planting requirements of modern pears due to the reasons of complex breeding genetic background, long childhood, incompatibility of self-breeding and the like of the pears. Therefore, breeders are constantly exploring a high-quality breeding method to breed new stress-resistant pear varieties. The rapid progress of the biotechnology and the tissue culture technology utilizes the genetic engineering means to culture the variety with strong resistance, and has wide application and development prospect for pear stress-resistant breeding.

Under the condition of salt stress, various physiological activities of plants are adversely affected, which is mainly reflected in the initial osmotic stress, so that the root system of the plant cannot absorb water or even lose water, the growth rate of the plant is reduced, and the plant can die; secondly, toxic substance accumulation and nutrient element deficiency caused by ion imbalance; then, the oxidative stress causes the generation and accumulation of active oxygen free radicals in plants, and the physiological metabolism is disturbed. During long-term evolution, plants develop a complex series of salt tolerance mechanisms to adapt to the external environment, including osmotic regulation mechanisms, antioxidant mechanisms, stress signal transduction, endogenous hormones, and changes in photosynthesis pathways.

According to the existing research, the survival of plant cells depends on the activity of a plant vacuole V-type proton pump (V-ATPase) under the stress conditions of salt, drought and the like, so that the V-ATPase has a close relationship with the stress resistance of plants (Dietz)&Arbinger, 1996). Effectively remove excessive Na⁺Discharge of cytoplasm and compartmentalization to vacuole are the major mechanisms by which plants adapt to salt stress. V-ATPase uses the energy released by ATP hydrolysis to transport protons from the cytoplasm to the vacuole, thereby creating an ion gradient to regulate the transfer of various ions and metabolites. When plants are stressed by salt, Na on vacuole can be activated by proton concentration gradient formed by hydrolysis of V-ATPase⁺/H⁺Antiporter protein, Na⁺/H⁺Reverse transport proteins by passing Na⁺Transporting the Na in the cells by a reverse concentration gradient⁺Is either excreted or compartmentalized into the vacuole, thus maintaining intracellular osmotic potential stability, reducing water efflux, avoiding water stress (chinnusmamy et al, 2006). Therefore, the salt tolerance of V-ATPase and plants is inseparable.

V-ATPase was originally identified as liquidNO on bubble film₃ ^-Sensitive ATP hydrolase. A great deal of information about the structure, function and regulation of plant V-ATPases has been accumulated during the course of ongoing research. V-ATPase (vacuolar ATPase) is an ATP-dependent proton pump (Stevens et al, 1997) located in the inner membrane of eukaryotic cells and in the plasma membrane of specialized cells. The structure of V-ATPase is complex and is a complex of over ten different subunits. Sequence information for all subunit V-atpases was obtained on arabidopsis and ice-leaf days until 2000 (Dietz et al, 2001). The V-ATPase polymerase contains two distinct domains: a transmembrane proton channel (V0) and an extracellular soluble domain (V1) (Diepholz et al, ZHao et al). V0 consists of subunits a, c, c', d and e, constituting a transmembrane proton channel. V1 has eight subunits, a-H, containing ATP binding sites, and functions as ATP hydrolysis and regulation. The two regions, V0 and V1, are tightly coupled and cleavage results in their loss of activity (Wang et al, 1999). Wherein the subunit B of the V-ATPase has a molecular weight of about 55-60 kDa, has a rather conserved nucleotide sequence, has two regulatory sites and participates in the formation of catalytic sites (Liu et al, 1996, Wang et al, 2011, Stevens)&Forgac,1997)。

There are many reports showing that V-ATPase activity is enhanced under salt treatment conditions. Ratajczak et al and Tsiantis et al suggest that the activity of each subunit of V-ATPase is enhanced under salt treatment conditions in ice-leaf sunflowers to accommodate salt stress, and that the B subunit has a coordinated expression pattern with the C subunit activity (Ratajczak et al, 1994, Tsiantis et al, 1996). Li et al found that under the salt treatment conditions, the expression level of the H subunit of V-ATPase in suaeda salsa was increased, so that the activity of V-ATPase was enhanced and Na was added⁺Transport into the vacuole provides the driving force (Li et al, 2004). Kirsch et al placed mature sugar beet leaf in 400mM salt solution found that the expression level of the C subunit of V-ATPase increased and that the expression levels of the other subunits of V-ATPase increased accordingly, and proposed that V-ATPase and vacuolar Na⁺/H⁺The mechanism of co-expression of antiporters (Kirsch et al, 1996). These results all indicate that under salt treatment conditions, plants are able to resist salt stress by modulating V-ATPase activity, therebyThereby improving the salt tolerance of the plants. Expression of homotypic genes in different tissues and organs under external stress conditions may affect V-ATPase activity in response to stress (Goldack)&Dietz,2001)。

In addition, a plurality of transgenic experiments also fully verify that the V-ATPase can improve the salt tolerance of plants. Wang et al obtained an over-expressed Arabidopsis plant by transferring the maize V-ATPase gene TaVB into Arabidopsis, and the over-expressed strain has enhanced V-ATPase activity and greatly improved salt tolerance (Wang et al, 2011). WANG et al, transfer the B, C and H subunits of V-ATPase into alfalfa to obtain salt and salt tolerant alfalfa plants (WANG et al, 2016). Zhou et al by mixing Na⁺/H⁺The reverse transport protein gene AtNHX1 is transferred into tobacco, the activity of the over-expressed strain V-ATPase is enhanced, the salt tolerance of the strain is enhanced, and Na is presumed to be⁺/H⁺Antiporters function synergistically with V-ATPase (Zhou et al, 2011). It has also been reported that V-ATPase activity is expressed by drought, cold stress, and acid stress (Dietz et al, 2001).

The birch-leaf pear is a stock which is commonly applied in the pear industry, has extremely high salt tolerance and is an ideal material for researching the salt tolerance of woody plants and cloning related salt-resistant genes. Therefore, cloning of the salt-resistant related gene of the pyrus betulaefolia is the key and the basis of salt-resistant gene engineering, but no report about the salt-resistant gene in the pyrus betulaefolia exists in the prior art.

Disclosure of Invention

In view of the above, the present invention aims to provide a birch-leaf pear vacuole type proton pump PbVHA-B1 and an application thereof in plant salt-resistant genetic improvement.

The invention provides a birch pear vacuole proton pump PbVHA-B1, the amino acid sequence of which is SEQ ID No. 1.

The invention provides a coding gene of a birch-leaf pear vacuole proton pump PbVHA-B1, the nucleotide sequence of which is SEQID No. 2.

The invention provides a primer pair for amplifying the coding gene, wherein the nucleotide sequence of an upstream primer in the primer pair is SEQ ID No. 3; the nucleotide sequence of the primer pair downstream primer is SEQ ID No. 4.

The invention provides a plant expression vector containing the coding gene.

Preferably, the basic vector in the plant expression vector is PCMBIA 1300;

the multiple cloning sites of the coding gene inserted into the PCMBIA1300 are XbaI and BamHI.

The invention provides application of the vacuole proton pump PbVHA-B1 in pyrus betulaefolia, the coding gene, the primer pair or the plant expression vector in salt-tolerant plant breeding or transgenic salt-tolerant plant cultivation.

Preferably, the salt comprises Na⁺Or K⁺。

Preferably, the concentration of the salt is not higher than 1000 mmol/L.

Preferably, the plant comprises arabidopsis thaliana or pyrus betulaefolia.

The invention provides a coding gene of a vacuole proton pump PbVHA-B1 in pyrus betulaefolia, and the nucleotide sequence of the coding gene is shown as SEQID NO. 2. The coding gene is introduced into a birch pear seedling, sampling is carried out at a corresponding time point under the treatment of 200mM NaCl, and the relative expression quantity of the coding gene is analyzed by adopting real-time quantitative PCR, so that the result shows that the relative expression quantity of the coding gene is gradually increased along with the prolonging of the treatment time of NaCl within 12h, which shows that the coding gene responds to salt stress treatment and has the function of regulating and controlling salt resistance.

The invention provides application of the vacuole proton pump PbVHA-B1 in the pyrus betulaefolia, the coding gene or the primer pair in salt-tolerant plant breeding or transgenic salt-tolerant plant cultivation. Respectively constructing an arabidopsis thaliana transformation vector and a gene silencing birch pear seedling transformation vector to obtain positive plant seedlings, and performing salt treatment on the positive plant seedlings, wherein the results show that: the conductivity of the transgenic arabidopsis positive plant seedling is obviously reduced compared with that of a Wild Type (WT), the chlorophyll content is obviously increased compared with that of the wild type, and Na in a transgenic line is⁺The content is lower, is 1/2 of wild type content, and K⁺The content is higher and is 2 times of the wild type content, the seed germination rate is improved by 38 percent to 34 percent compared with the wild type, and the root length is longer than the wild typeThe growth type is increased by 17.3-38.2%, which shows that the positive plant seedlings have stronger salt tolerance than wild plants. Virus silencing of Hydrogen peroxide (H) in birchleaf pear seedling Positive plants₂O₂) And superoxide anion (O)₂ ^-) The content activity of the compound is higher than that of a wild type, the active oxygen residue in a plant body is higher, and the cell damage is larger. These results indicate that the overexpressed PbVHA-B1 coding gene is capable of efficiently maintaining intracellular Na⁺/K⁺The concentration balance keeps the cell osmotic potential stable, so that the plant can better adapt to salt stress, and the silencing of the PbVHA-B1 encoding gene reduces the active oxygen scavenging capability of the plant, so that the salt tolerance of the plant is weakened.

Drawings

FIG. 1 is a schematic technical flow chart of the construction method of transgenic salt-tolerant plants according to the present invention;

FIG. 2 is a schematic diagram showing the expression of the gene encoding PbVHA-B1 under salt, dehydration, abscisic acid (ABA) and low-temperature stress in example 2; wherein, FIG. 2-A shows that the coding gene is sampled at corresponding time points under the treatment of birch pear seedlings (not transgenic) in a 200mM NaCl solution, and the relative expression quantity of the coding gene is analyzed by adopting real-time quantitative PCR; FIG. 2-B is an expression pattern of dehydration of a seedling of Pyrus betulaefolia at room temperature without a time point; FIG. 2-C shows the relative expression level of the gene of the present invention analyzed by real-time quantitative PCR from the birch pear seedlings sampled at corresponding time points under the treatment of 100. mu. MABA; FIG. 2-D shows the relative expression level of the coding gene analyzed by real-time quantitative PCR from birch-leaf pear seedlings sampled at corresponding time points under low temperature treatment at 4 ℃;

FIG. 3 is a diagram of a plant entity of PbVHA-B1 transgenic Arabidopsis in example 3, FIG. 3-A is a diagram of electrophoresis result of constructed recombinant vector, FIG. 3-B is a diagram of PCR identification of transgenic plant of Arabidopsis T0 generation using gene specific primer, wherein M: marker, +: plasmid, -: wild type plant, 1-8: transgenic lines, FIG. 3-C is overexpression analysis of RT-PCR identified transgenic Arabidopsis TI generation plants;

FIG. 4 shows the transient transformation of seedlings of Du pear silenced by PbVHA-B1 virus in example 4, FIG. 4-A shows the vector construction, and FIG. 4-B shows the real-time quantitative PCR analysis of the expression level of the coding gene of PbVHA-B1 in different virus-silenced strains of autumn pear;

FIG. 5 shows the results of determination of phenotype and physiological index before and after treatment with Wild Type (WT) sodium chloride and the gene line encoding PbVHA-B1 in example 5, wherein FIG. 5-A shows the phenotype of 20-day-old Arabidopsis plants before treatment with 200mM sodium chloride for 15 days; FIG. 5-B is a photograph of fluorescent chlorophyll from 20-day-old Arabidopsis plants before treatment with 200mM sodium chloride for 15 days; FIG. 5-C is the phenotype of a 20-day-old Arabidopsis plant after 15 days of 200mM sodium chloride treatment; FIG. 5-D is a photograph of fluorescent chlorophyll of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days; FIG. 5-E is a phenotypic picture of 7-day-old Arabidopsis plants after treatment on MS medium containing 100mM sodium chloride for 7 days; FIG. 5-F is a statistical result of root length of 7-day-old Arabidopsis plants treated on MS medium containing 100mM sodium chloride for 7 days; FIG. 5-G are pictures of phenotypes of 7-day-old Arabidopsis plants after 5-day treatment on MS medium containing 150mM sodium chloride; FIG. 5-H is a graph of statistics of root length of 7-day-old Arabidopsis plants treated for 5 days on MS medium containing 150mM sodium chloride; FIG. 5-I is the germination of Arabidopsis seeds grown for 6 days on MS medium with 75mM sodium chloride; FIG. 5-J are statistical plots of germination rates of Arabidopsis seeds grown for 6 days on MS medium with 75mM sodium chloride; FIG. 5-K is the germination of Arabidopsis seeds grown for 6 days on MS medium with 100mM sodium chloride; FIG. 5-L is a graph showing statistics of germination rates of Arabidopsis seeds grown for 6 days on MS medium containing 100mM sodium chloride; FIG. 5-M is a graph showing chlorophyll extraction results of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days; FIG. 5-N is a graph showing chlorophyll determination results of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days; FIG. 5-O is a graph showing the results of conductivity measurement of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days;

FIG. 6 is histochemical staining analysis H of the gene encoding PbVHA-B1 in Arabidopsis thaliana of example 5₂O₂And O₂ ^-Accumulation, FIGS. 6-A and 6-B show that 20-day-old Arabidopsis plants were treated with 200mM sodium chloride for 7 days before and after the untransformed plants and three transgenic lines were subjected to reactive oxygen histochemical staining using MirabilitumH is respectively treated by base tetrazole (NBT) and Diaminobenzidine (DAB)₂O₂(FIG. 6-A) and O₂ ^-(FIG. 6-B) dyeing; FIGS. 6-C and 6-D are the treatment of 7-day-old Arabidopsis plants on MS minimal medium containing 100mM sodium chloride for 3 days, and then the untransformed plants and three transgenic lines were treated with Nitro-tetrazole (NBT) and Diaminobenzidine (DAB), respectively, on H₂O₂(FIG. 6-C) and O₂ ^-(FIG. 6-D) dyeing;

FIG. 7 shows Na in Arabidopsis thaliana cells transformed with PbVHA-B1 gene of example 5⁺And K⁺Content determination, FIG. 7-A shows Na content of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days⁺Content comparison graph, FIG. 7-B is K after 200mM sodium chloride treatment for 15 days for 20-day-old Arabidopsis plants⁺FIG. 7-C is a graph showing Na content of 20-day-old Arabidopsis plants treated with 200mM sodium chloride for 15 days⁺/K⁺A content comparison graph;

FIG. 8 is a graph of phenotypic and physiological assignment mapping of PbVHA-B1 encoding gene-silenced seedling lines of Du pear (OE1, OE2 and OE3) and wild-type plants (WT) incubated for 20 days with 200mM sodium chloride in example 6, wherein FIG. 8-A is a phenotype map of a potted plant growing normally (before treatment), FIG. 8-B is a phenotype map of 20 days of 200mM sodium chloride watering, FIG. 8-C is a graph showing the results of conductivity measurement after 20 days of pouring with 200mM sodium chloride, FIGS. 8-D and 8-E are a graph showing the results of chlorophyll measurement after 20 days of pouring with 200mM sodium chloride (FIG. 8-D) and the results of chlorophyll extraction (FIG. 8-E), FIG. 8-F is a photograph of fluorescent chlorophyll from potted plants 20 days before watering with 200mM sodium chloride, FIG. 8-G is a photograph of fluorescent chlorophyll from potted plants 20 days after watering with 200mM sodium chloride;

FIG. 9 histochemical staining analysis of Du pear seedlings encoding gene silencing of PbVHA-B1H in example 6₂O₂And O₂ ^-FIG. 9-A and FIG. 9-B are graphs of accumulation results of activated oxygen histochemical staining of wild type and three gene silencing strains of Du pear seedlings before and after 10 days of 200mM sodium chloride treatment with Nitrotetrazole (NBT) and Diaminobenzidine (DAB) for H₂O₂(FIG. 9-A) and O₂ ^-FIG. 9-B is a graph showing the results of staining.

Detailed Description

The invention provides a birch pear vacuole membrane proton pump PbVHA-B1, the amino acid sequence of which is shown in SEQ ID NO.1

(MAVSQNNHDMDEGNLEVGMEYRTVSGVAGPLVILEKVKGPKFQEIVNI RLGDGTTRRGQVLEVDGEKAIVQVFEGTSGIDNKYTTVQFTGEVLKTPVS LDMLGRIFNGSGKPIDNGPPILPEAYLDISGSSINPSERTYPEEMIQTGISTID VMNSIARGQKIPLFSAAGLPHNEIAAQICRQAGLVKRLEKSESLLDAGDV EDDNFAIVFAAMGVNMETAQFFKRDFEENGSMERVTLFLNLANDPTIERII TPRIALTTAEYLAYECGKHVLVILTDMSSYADALREVSAAREEVPGRRGYP GYMYTDLAQIYERAGRIEGRKGSITQIPILTMPNDDITHPTPDLTGYITEGQ IYIDRQLHNRQIYPPINVLPSLSRLMKSAIGEGMTRRDHSDVSNQLYANYA IGKDVQAMKAVVGEEALSSEDLLYLEFLDKFEKKFVSQGAYDTRNIFQSL DLAWTLLRIFPRELLHRIPAKTLDLFYSRDAAN). The PbVHA-B1 encodes 490 amino acids, has an isoelectric point of 4.98 and a molecular weight of 54.47 KDa. The PbVHA-B1 has the function of promoting proton transport and maintaining plant cell Na⁺/K⁺Balance and can increase the ROS resistance of the carrier plant, thereby indicating that the gene has the function of salt resistance. In the invention, the PbVHA-B1 is obtained by recombinant expression of a gene encoding the PbVHA-B1. The method of the recombinant expression is not particularly limited, and a recombinant expression method well known in the art may be used.

The invention provides a coding gene of the birch pear vacuole proton pump PbVHA-B1, the nucleotide sequence of which is shown as SEQID NO.2

(atggctgtttcacaaaacaatcatgacatggacgagggaaacctagaggtcggaatggaatacagaactgtgtct ggtgtggccggacctctggttatccttgaaaaagttaagggacctaagtttcaagagattgttaacattcgtttgggag atggaacaactcgacgtggtcaagtcctggaagttgatggagagaaagctattgtacaggttttcgaaggaacatctg gaattgacaacaagtacactactgtgcaattcacaggagaggttttgaaaactccagtctcacttgacatgcttgggcg catctttaatggctctgggaagcccattgataatggcccccctattttgcctgaggcttacctagacatatctgggagtt ctattaatccgagtgagagaacatatcctgaagaaatgattcagactggaatttctactattgatgtcatgaactccattg cgagaggacaaaaaatcccccttttctctgctgctggtcttcctcataatgaaatagctgctcagatatgtcgccaggc cggtttggtcaagcggttggagaaatctgagagtcttcttgacgctggggacgtagaagacgacaactttgccattgt gtttgcagctatgggagtaaatatggagactgcacagttctttaagcgtgattttgaggaaaatggttcaatggagaga gtgaccctttttctgaatctggcaaatgaccctacaattgaacgcattattactcctcgtattgctcttactactgcagaat atttggcatatgaatgtgggaagcatgttcttgtcattctcactgatatgagttcttatgctgatgctcttcgtgaggtgtct gctgcccgagaggaagtgccgggaaggcgtggataccctgggtacatgtatactgatctggcacaaatctatgagc gtgctggaagaattgaagggcgaaaaggctctattacccaaattccgatcttaactatgccaaatgatgatattaccca ccccactccagatcttactggatatattactgagggacagatatacattgacaggcagctccacaacagacagatata tccaccaatcaatgtcctcccatcactatctcgtctgatgaagagtgctattggtgaaggcatgactcgccgggatcat tctgatgtatcaaatcagttatatgcaaattatgctattgggaaggatgtccaggcaatgaaagctgtggtcggagaag aagcactttcttcggaggacttgctatacctggagttcttggacaaatttgagaagaagtttgtgtcccaaggagcctatgacacccgtaacatcttccagtccctcgatttggcatggacgttgctgcgaatcttcccccgtgagcttctccaccgtat acctgcaaagacccttgacctgttctacagcagagatgcagctaattga). The source of the coding gene is preferably obtained by cloning.

The invention provides a primer pair for cloning the coding gene, which comprises a forward primer and a reaction primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.3 in a sequence table (ATGGCTGTTTCACAAAACAATCA); the nucleotide sequence of the reverse primer is shown as SEQ ID NO.4 in the sequence table (TCAATTAGCTGCATCTCTGCTGTA). The source of the primer pair is not particularly limited, and it may be synthesized by biosynthetic companies well known in the art. In the examples of the present invention, the primer pair was synthesized by Shanghai Bioengineering Co., Ltd.

The invention provides a plant expression vector containing the coding gene. The construction method of the plant expression vector comprises the steps of cloning the coding gene, carrying out enzyme digestion on the coding gene and the basic vector by adopting the same restriction enzyme, connecting the obtained coding gene sequence segment with the linear basic vector, and screening to obtain the plant expression vector. The basic vector in the plant expression vector is preferably PCMBIA 1300; the multiple cloning sites of the coding gene inserted into the PCMBIA1300 are XbaI and BamHI.

The invention provides application of the birch-leaf pear vacuole proton pump PbVHA-B1, the coding gene or the coding gene pair cloned by the primer pair in improving the salt tolerance of plants.

In the present invention, the application preferably comprises the steps of: and (3) recombining and expressing the coding gene amplified by PCR (polymerase chain reaction) or the primer in a plant to obtain the recombinant plant with salt tolerance (see figure 1). In the invention, the birch pear vacuole proton pump PbVHA-B1 or the coding gene amplified by the primer pair is silenced in plants, and the salt tolerance of the obtained recombinant expression plants is weakened.

In the present invention, the reaction procedure of the PCR amplification is preferably pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles. The coding gene is preferably transformed into plants in the form of a p1300-PbVHA-B1 recombinant vector and silenced in plants in the form of a pTRV2-PbVHA-B1 recombinant vector. The construction method of the p1300-PbVHA-B1 and pTRV2-PbVHA-B1 recombinant vectors is not particularly limited in the present invention, and construction methods known in the art can be adopted.

The method for the recombinant expression and silencing of the coding gene or the coding gene amplified by the primer pair through PCR in the plant preferably adopts an agrobacterium-mediated genetic transformation method. The method for said agrobacterium-mediated genetic transformation is not particularly limited in the present invention, and conventional methods well known in the art may be used. The plants preferably include arabidopsis and pyrus betulaefolia.

In the present invention, before the salt tolerance of the recombinant expression plants and the gene silencing plants is tested, the screening of positive plants is preferably included. The screening method of the positive transgenic plant is preferably carried out by adopting a PCR amplification method. During screening, a forward primer of the primer for PCR amplification has a nucleotide sequence shown as SEQ ID NO.3 in a sequence table; the primer reverse primer for PCR amplification has a nucleotide sequence shown as SEQ ID NO.4 in a sequence table. The reaction procedure for the PCR amplification is as follows:

the PCR reaction system for PCR amplification is as follows:

reaction components	Dosage (mu l)
		Template DNA	1
PCR buffer solution	2
		dNTDMix(2.5mMol/L)	1.6
Forward primer	1
		Reverse primer	1
TaqDNA polymerase (5U)	0.2
		Ribozyme-free water	13.2

After the PCR amplification is finished, if the plant line to be detected can amplify a fragment (1473bp) with an expected size, the result shows that the test plant is a positive transgenic line.

The screening method of the positive gene silencing plant is preferably carried out by adopting a method of checking the gene expression quantity of the positive gene silencing plant by qRT-PCR. During screening, a forward primer of a primer used in qRT-PCR has a nucleotide sequence (TCCTCGGATTCCATTGCCCAGC) shown as SEQ ID NO.5 in a sequence table; the primer reverse primer for PCR amplification has a nucleotide sequence (CACAAGAACAGTTTCCTTGGTTTTC) shown as SEQ ID NO.6 in a sequence table; the internal reference primer Tublin has a nucleotide sequence shown as SEQ ID NO.7(TGGGCTTTGCTCCTCTTAC) and SEQ ID NO.8 (CCTTCGTGCTCATCTTACC) in the list.

The real-time quantitative PCR reaction procedure was as follows:

the real-time quantitative PCR reaction system comprises the following steps:

reaction components	Dosage (mu l)
		Template cDNA	0.1
Forward primer	0.2
		Reverse primer	0.2
2 × SYBR fluorescent primer	5
		Ribozyme-free water	4.5

After the real-time quantitative PCR amplification is finished, if the expression quantity of the gene amplified by the plant line to be detected is obviously lower than that of the gene in the wild plant, the plant line to be detected is a positive silent plant line.

In the invention, the obtained positive transgenic line and gene silencing line are subjected to salt treatment, and the phenotype, physiological index and active oxygen of the positive transgenic line and gene silencing line obtained after the treatment are determined to verify the salt tolerance function of the positive transgenic line and gene silencing line. The salt species preferably includes Na⁺And K⁺. The concentration of the salt is not higher than 1000mmol/L, more preferably 800-100 mmol/L, further preferably 200-600 mmol/L, and most preferably 500 mmol/L. The Na is⁺And K⁺The measuring method is preferably flame photometer measurement. Through Na⁺And K⁺The measurement result shows that: na in transgenic lines compared to wild type Arabidopsis plants⁺The content is lower, is 1/2 of wild type content, and K⁺The content is higher and is 2 times of the wild type content, which shows that the positive transgenic line can better absorb K from the outside⁺And excess Na in cytoplasm⁺Excreted and partitioned into vacuole to maintain Na in cytoplasm⁺/K⁺The plant growth regulator is relatively stable, adjusts the osmotic balance of cells, and avoids the damage of salt stress to plants.

In the present invention, the method for measuring the active oxygen is preferably a method for measuring the leaf H of a plant by using DAB and NBT histochemical staining method₂O₂And O₂ ^-Accumulation was analyzed (by the shade and extent of staining), visually observed and photographed. The active oxygen includes hydrogen peroxide (H) respectively₂O₂) And superoxide anion (O)₂ ^-). The method is found by measuring the active oxygen content change of a positive transgenic line and a wild type arabidopsis plant: after 7 days of salt stress, with DAB stained leaves, the wild type appeared dark brown and the stained leaf area was significantly larger than the transgenic lines, and with NBT stained leaves, the wild type line was darker in blue and larger in area than the transgenic line, indicating that the positive transgenic line had a lower ROS (H) than the wild type under salt stress (H)₂O₂And O₂ ^-) Accumulate, thereby ensureLess cell damage is evidenced. The active oxygen content change of the positive gene silencing strain and the wild type birch pear seedling is detected by detecting that: after 7 days of salt stress, the leaf area and color depth of the leaves stained with DAB and NBT were significantly greater in the gene silencing lines than in wild type Pyrus betulaefolia seedlings, indicating that the gene silencing encoded by PbVHA-B1 results in ROS (H) in plant cells₂O₂And O₂ ^-) The excessive accumulation of the plant salt-tolerant plant causes more serious damage to cell membranes and reduces the salt-tolerant capability of the plant.

In the present invention, the conductivity measurement and the chlorophyll extraction and measurement method are not particularly limited, and those well known in the art may be used. Analyzing the measured results of the conductivity and the chlorophyll, and finding that: the conductivity of the positive transgenic plant seedling is obviously reduced compared with that of a Wild Type (WT), the chlorophyll content is obviously increased compared with that of the wild type, the seed germination rate is improved by 38-34% compared with that of the wild type, and the root length is increased by 17.3-38.2% compared with that of the wild type, which shows that the positive transgenic plant seedling has stronger salt resistance than that of the wild type. On the contrary, the conductivity of the gene silencing positive seedling is obviously increased compared with the Wild Type (WT) and the chlorophyll content is obviously reduced compared with the wild type, which shows that the salt resistance of the silencing positive seedling is weaker than that of the wild type.

The present invention is not particularly limited in kind of the plant, and is applicable to any plant, such as Arabidopsis thaliana or Pyrus pyrifolia.

The present invention provides a birch-leaf pear vacuole type proton pump PbVHA-B1 and its application in plant salt-resistant genetic improvement, which are described in detail in the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Cloning method of birchleaf pear PbVHA-B1 gene full-length cDNA

A vacuole proton pump PbVHA-B1 was selected from Du pear, primers were designed based on the sequence of PbVHA-B1 gene and primerpremier 5.0, and the full length was amplified from Du pear by RT-PCR. The detailed steps are as follows: first strand cDNA synthesis was performed according to the protocol of the TIANGEN reverse transcription kit. The obtained first strand cDNA was used for the PbVHA-B1 geneThe PCR amplification of (1). The total reaction volume of PCR amplification is 50 mu l, wherein 1 mu l of pyrus betulaefolia cDNA, 2.5 mu l of each of upstream and downstream primers, 1 mu l of Taq DNA polymerase, 25 mu l of Buffer solution and sterilized ddH₂O18. mu.l. The reaction procedure for PCR amplification was as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles. After the PCR product which generates a single band after the amplification is subjected to 1% agarose gel electrophoresis, cutting off a target band, and recovering the specific target band according to the steps of the instructions of the gel recovery kit.

The recovered and purified product is connected with a pEASY-Bluntzero vector, and the molar ratio of the gene to the vector in the connection system is 3: 1. The total reaction volume was 5. mu.l, 4.5. mu.l of PCR-purified product, 0.5. mu.l of vector, ligated for 30min at 25 ℃ and transformed into E.coli competent DH 5. alpha. by heat shock, PCR-verified and sequenced with the gene sequence primers of interest (completed by Shanghai Biotechnology Limited). The sequencing result is shown as SEQ ID NO.2, and the amino acid sequence obtained by codon conversion is shown as SEQ ID NO. 1.

Example 2

qRT-PCR analysis of PbVHA-B1 encoding gene under different stress conditions

In order to analyze the response pattern of the PbVHA-B1 gene in pyrus betulaefolia to high salt, dehydration, abscisic acid (ABA) and low temperature treatment, the expression pattern of the PbVHA-B1 encoding gene was analyzed using Real-time PCR technology. The material selected in the experiment is the birch pear seedling, and the birch pear seedling is cultured in the national pear industry and technology research center of Nanjing agriculture university. Selecting 45-day-old birch pear seedlings with consistent growth conditions and good health conditions, respectively carrying out different stress treatment, sampling at corresponding time points, quickly freezing the samples by using liquid nitrogen, and storing in an ultra-low temperature refrigerator at minus 80 ℃. Salt treatment, seedlings treated with 200mM NaCl solution were sampled at 0, 1, 6, 12, 24 and 48 h; dewatering, namely placing the seedlings on dry filter paper, and dewatering at room temperature (relative humidity is 60-70%) for 0, 6, 9, 12 and 24 hours for sampling; abscisic acid (ABA) treatment, seedlings treated with a solution of 100. mu. MABA were sampled at 0, 6, 9, 12 and 36 h; low-temp. treatment, placing the seedlings in 4 deg.C incubatorSamples were taken at 0, 3, 9, 12, 24 and 72h treatment RNA was extracted using the plant Total RNAI Kit Plus from Dow-Co-Ltd, and first strand DNA synthesis was performed with reference to the manual of the TANGEN reverse transcription kit.10. mu.l of a reaction system was 5. mu.l of 2 × SYBRPremixExTaq, 0.1. mu.l of cDNA, 0.4. mu.l of primers (SEQ ID NO.5 and SEQ ID NO.6), and 4.5. mu.l of ddH₂And O. The reaction program of real-time quantitative PCR using Tublin as internal reference primer (sequence of primer pair is SEQ ID NO.7 and SEQ ID NO.8) is shown in Table 1.

TABLE 1 real-time quantitative PCR procedure

The results of the qRT-PCR are shown in FIG. 2. FIG. 2 is a schematic diagram of the expression of the PbVHA-B1 encoding gene under high salt, dehydration and ABA stress, wherein FIG. 2-A is a corresponding time point sampling of the encoding gene under the treatment of birch pear seedlings (not transgenic) in 200mM NaCl solution, and the relative expression of the gene is analyzed by real-time quantitative PCR; FIG. 2-B is an expression pattern of dehydration of a seedling of Pyrus betulaefolia at room temperature at different time points; FIG. 2-C shows the relative expression of the coding gene of the seedlings of Pyrus betulaefolia, which were sampled at corresponding time points under the treatment of 100. mu. MABA solution, and analyzed by real-time quantitative PCR; FIG. 2-D shows the relative expression of the coding gene in the seedlings of Pyrus betulaefolia after being treated at 4 ℃ and sampled at the corresponding time points by real-time quantitative PCR. As can be seen from FIG. 2-A, the expression level of the coding gene showed an increasing tendency within 12 hours as the treatment time was prolonged, and the expression level began to decrease after 12 hours. This shows that the coding gene is affected by salt stress, so that the salt-resistant mechanism is responded, and the salt-resistant function of the pyrus betulaefolia is realized. FIG. 2-B shows that the expression level of PbVHA-B1 gene increased and reached the highest level within 9 hours after dehydration treatment. FIG. 2-C shows that the expression level of PbVHA-B1 gene was lowest at 0h, highest at 12h and then decreased during ABA treatment. FIG. 2-D shows that after low-temperature treatment, the expression level of the PbVHA-B1 gene is increased continuously within 9h and is reduced within 9-72 h. These results all indicate that the expression level of PbVHA-B1 gene is induced by many stresses.

Example 3

Genetic transformation of Arabidopsis thaliana

1. Construction of plant transformation vectors

Based on the multiple cloning site of the PCMBIA1300 vector and the coding region sequence of the gene encoding PbVHA-B1, restriction sites XbaI and BamHI were added, and the upstream and downstream primers (SEQ ID NO.9, GAGAACACGGGGGACTCTAG AATGGCTGTTTCACAAAACAATCA and SEQ ID NO.10, GCCCTTGCTC ACCATGGATCCATTAGCTGCATCTCTGCTGTAGAA) were designed using primerprimer 5.0 software according to the general principle of primer design. PCR amplification was performed using a clone of the gene encoding PbVHA-B1 as a template. The annealing temperature of PCR amplification is 58 ℃, and the PCR reaction system and the amplification procedure are the same as those of the PbVHA-B1 gene clone. After amplification, gel purification and recovery are carried out. The volume of the double digestion reaction of the PCMBIA1300 vector is 40 mu l, wherein: 10. mu.l of vector plasmid containing PCMBIA1300, 4. mu.l of 10 XM buffer, 1. mu.l each of XbaI and BamHI, and 24. mu.l of double distilled water. And (3) placing the mixture at 37 ℃ for enzyme digestion for 3-4 h, and then purifying and recovering the product. Adding the PbVHA-B1 encoding gene and the vector PCMBIA1300 into a ligation reaction system at a molar ratio of 2:1, wherein the reaction total volume is 10 mu l, and the reaction system comprises: 10 x buffer 1 mul, DNA recombinase 1 mul, double-enzyme-digested recovered PbVHA-B1 gene 4 mul, double-enzyme-digested recovered PCMBIA1300 vector product 2 mul, double-distilled water 2 mul, reacting at 37 ℃ for 30min to obtain ligation product. The ligation product was transformed into E.coli DH 5. alpha. and cultured for 16h in LB solid plates containing 50mg/L kanamycin. And (3) selecting points of the screened positive clones, shaking the positive clones, extracting plasmids for PCR identification, sequencing to determine that no coding frame mutation exists, obtaining recombinant clones containing the inserted target fragments, naming the recombinant clones as p1300-PbVHA-B1 recombinant vectors, and introducing the recombinant vectors p1300-PbVHA-B1 into agrobacterium GV3101 by using a freeze-thaw method.

2. The agrobacterium-mediated genetic transformation of arabidopsis thaliana was as follows:

(1) and (3) agrobacterium culture: taking agrobacterium tumefaciens bacterial liquid stored in an ultra-low temperature refrigerator, streaking the agrobacterium tumefaciens bacterial liquid on a flat plate added with LB (lysogeny broth) with 50mg/L kanamycin and 50mg/L rifampicin, putting the streaked bacterial liquid on an incubator with the temperature of 28 ℃ for culturing for 36-48 h, scraping streaked bacterial plaque, adding liquid MS (2.37g/LMS +50g/L sucrose +0.1mg/L IBA, pH value 5.8) into a culture medium, rotating for 30min at the temperature of 28 ℃ for shaking culture, and adding a surfactant silwet77200 mu L/L for infection when the bacterial liquid concentration reaches OD (OD) of 0.8-1.0.

(2) Infection: taking a wild type arabidopsis thaliana plant with a stem height of about 10cm, removing all seed pods, then pouring into a glass bottle containing the prepared agrobacterium tumefaciens bacterial liquid, vacuumizing, maintaining the pressure of 0.05Mpa for 5min, and placing for 24h away from the sun.

(3) Culturing: the plants were grown to seed and mature seeds were harvested by conventional methods (T0 generation).

3. Screening for transgenic Positive seedlings

Obtaining seeds of T0 generations of Arabidopsis thaliana transformed with PbVHA-B1 gene according to the method, sterilizing the surfaces of the seeds of T0 generations, uniformly spreading the seeds on an MS selective culture medium containing 50mg/L hygromycin and 50mg/L timentin, placing the seeds in a 22 ℃ illumination 16h/d for culture, selecting plants with fast growth and long root growth after one week of growth, transplanting the plants into sterilized nutrient soil, and growing for a period of time.

3.1 transgenic Arabidopsis DNA extraction method

Obtaining the PbVHA-B1 transgenic tobacco according to the method, extracting DNA from each arabidopsis thaliana, and designing a primer gene inner primer to carry out PCR amplification to identify positive seedlings.

(1) An appropriate amount of Arabidopsis thaliana leaves was ground to powder with liquid nitrogen, and then 500. mu.l of 65 ℃ preheated CTAB (100mmol/L Tris-HCl solution pH 8.0, 1.5mmol/L NaCl, 50mmol/L EDTApH 8.0, 2% w/v CTAB, 65 ℃ water bath dissolved sufficiently for use) and 10. mu.l of beta-mercaptoethanol were added and mixed well.

(2) Heating in 65 deg.C water bath for 30min, taking out every 10min, slightly turning upside down, and mixing; centrifuging at 10000g for 10min at normal temperature; collecting supernatant, adding 500 μ l chloroform isoamyl alcohol (chloroform: isoamyl alcohol volume ratio is 24: 1), reversing and mixing;

(3) centrifuging at 10000g for 10min, collecting supernatant 450 μ l to a new 1.5ml centrifuge tube, adding isopropanol 450 μ l, and mixing by turning upside down;

(4)10000g, centrifuging for 10min, discarding the supernatant, rinsing with 1mL of 75% ethanol water solution for 2 times, 10000g, centrifuging for 10min, completely removing ethanol, and placing on a superclean bench for ventilation drying until the DNA is colorless and transparent;

(5) adding 50 μ l of ultrapure water, placing in an incubator at 65 ℃ for dissolving for 40min, and performing gel detection.

3.2 Positive transgenic plant detection

PCR amplification is carried out by using primer gene specific primers. The reaction procedures and systems are shown in tables 3 and 4, respectively. PCR was performed using the forward and vector reverse primers (SEQ ID NO. 11: ATGGCTGTTTCACAAAA CAATCA and SEQ ID NO. 12: CGTCGTCCTTGAAGAAGATG) to amplify fragments of the expected size in the selected transgenic lines, indicating that they were positive transgenic lines.

TABLE 3 PCR reaction procedure

TABLE 4 PCR reaction System

Reaction components	Dosage (mu l)
		Template DNA	1
PCRBuffer	2
		dNTDMix(2.5mMol/L)	1.6
Right forward primer	1
		Left reverse primer	1
TaqDNA polymerase (5U)	0.2
		Ribozyme-free water	13.2

Expression of PbVHA-B1 in Arabidopsis was mediated by Agrobacterium. Through molecular genetic analysis, the transgenic arabidopsis thaliana with single copy homozygous insertion and stably expressing PbVHA-B1 from T1-T2 generations is identified, so that the phenotypic character can be stably inherited. Insertion site analysis demonstrated that the phenotypic changes of the respectively transgenic PbVHA-B1 material were not caused by transgenic manipulation affecting other genes. Therefore, the transgenic material provides material guarantee for the research of the project. Vector construction as shown in FIG. 3-A, transgenic plants of T0 generation identified by PCR as shown in FIG. 3-B, and transgenic plants of T1 generation identified by RT-PCR as shown in FIG. 3-C, wherein #1, #5 and #6 are three overexpression lines.

Example 4

Transient transformation of birchleaf pear seedlings

1. Construction of Virus-induced Gene silencing vector

Viral silencing vectors were constructed according to the method of example 3. The double enzyme cutting sites of the viral silencing vector pTRV2 are XbaI and SacI, the coding gene of PbVHA-B1 is amplified and inserted into the middle of the two enzyme cutting sites on the vector by using a primerprimer 5.0 software to design an upstream primer and a downstream primer (SEQ ID NO.13: AAGGTTACCGAATTCTCTAGAATGGCT GTTTCACAAAACAATCA and SEQ ID NO.14: GGCCTCGAGACGCGTGAGC TCTCAATTAGCTGCATCTCTGCTGTA) according to the general principle of primer design, and the recombinant vector pTRV2-PbVHA-B1 is obtained and is transformed into the competence of agrobacterium GV 3101.

2. Virus-induced gene silencing of birch pear seedlings

(1) And (3) agrobacterium culture: taking the agrobacterium tumefaciens bacterial liquid stored in an ultra-low temperature refrigerator, and carrying out shake culture for 12h in LB liquid culture medium containing 50mg/L of kanamycin and 50mg/L of rifampicin at 28 ℃ and 220 rpm. Will be culturedThe resulting bacterial solution was centrifuged at 6000g for 10min to collect the cells, which were then treated with an infecting solution (10mM MgCl. solution)₂10mM MES and 200mM acetosyringone, pH value 5.6) is added, and the precipitate is resuspended until the OD reaches 0.8-1.0;

(2) inducing with bacterial liquid: placing the bacteria liquid with the well adjusted OD value in dark condition, and inducing at 100rpm normal temperature for 4 h;

(3) pear seedling injection: the control group was prepared by mixing pTRV1 and pTRV2 bacterial solutions at a ratio of 1:1, and the experimental group was prepared by mixing pTRV1 and pTRV2-PbVHA-B1 bacterial solutions at a ratio of 1:1, and the seedlings of birch pear seedlings which were aged 45 days and had consistent growth and good health were injected.

3. Identification of virus-induced gene silencing inhibition positive vaccine

And (3) carrying out dark treatment on the pear seedlings after injection at normal temperature for 12h, then recovering normal culture for 5 days, independently sampling each strain, extracting RNA of pear seedling samples of a control group and an experimental group, detecting the complete structure of the pear seedling samples by glue running, adjusting the total amount of the RNA to be 3 mu g, carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid) after the concentration of the RNA is determined by using Nanodrop (the concentration of the RNA is 200-1000 ng/mu l), and carrying out qPCR (quantitative polymerase chain reaction) amplification by using Tublin genes of pears as reference genes. The nucleotide sequence of the Tublin primer is as follows:

tublin forward primer: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO. 7);

tublin reverse primer: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID NO. 8).

The brightness of bands amplified by Tublin is consistent, which indicates that the concentration of reverse transcription cDNA is the same, then a PbVHA-B1 specific primer and a pear internal reference primer Tublin are used for qRT-PCR detection, the expression quantity of a strain to be detected is analyzed, and the nucleotide sequence of the PbVHA-B1 specific primer is as follows:

a forward primer: 5'-TCCTCGGATTCCATTGCCCAGC-3' (SEQ ID NO. 5);

reverse primer: 5'-CACAAGAACAGTTTCCTTGGTTTTC-3' (SEQ ID NO. 6).

According to the expression level of the PbVHA-B1 gene, three plants with lower expression levels are selected as virus-sinking-positive strains, which are named as OE1, OE2 and OE 3.

The results are shown in FIG. 4. FIG. 4-A is a gel electrophoresis chart of RNA extracted from pear seedling samples of a control group and an experimental group after transient injection; FIG. 4-B shows the qRT-PCR detection of gene expression level of positive plants with gene-specific primers and internal reference primer Tublin. This indicates that the PbVHA-B1 gene of virus-silenced birch pear seedling positive lines is silenced.

Example 5

PbVHA-B1 transgenic resistant plant resistance identification

1. Salt resistance analysis of transgenic Arabidopsis plants

To identify whether the PbVHA-B1 transgenic arabidopsis thaliana is associated with salt stress resistance, the control and transgenic lines were subjected to short-term salt stress and long-term salt stress. The PbVHA-B1 transgenic lines (#1, #5, #6) and Wild Type (WT) seeds received from the same batch were sterilized, sown on MS screening medium and commonly used MS non-resistant medium, respectively, and transplanted into soil for culture about 3 days after germination. Transgenic plants with different seedling ages are subjected to salt treatment, the treated phenotype is observed, the germination rate is counted, and the conductivity, chlorophyll, root length and the like are measured.

FIG. 5 shows the results of determination of phenotype and physiological index before and after treatment with Wild Type (WT) sodium chloride and the gene line encoding PbVHA-B1 in example 5, wherein FIG. 5-A shows the phenotype of 20-day-old Arabidopsis plants before treatment with 200mM sodium chloride for 15 days; FIG. 5-B is a photograph of fluorescent chlorophyll from 20-day-old Arabidopsis plants before treatment with 200mM sodium chloride for 15 days; FIG. 5-C is the phenotype of a 20-day-old Arabidopsis plant after 15 days of 200mM sodium chloride treatment; FIG. 5-D is a photograph of fluorescent chlorophyll of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days; FIG. 5-E is a phenotypic picture of 7-day-old Arabidopsis plants after treatment on MS medium containing 100mM sodium chloride for 7 days; FIG. 5-F is a statistical result of root length of 7-day-old Arabidopsis plants treated on MS medium containing 100mM sodium chloride for 7 days; FIG. 5-G are pictures of phenotypes of 7-day-old Arabidopsis plants after 5-day treatment on MS medium containing 150mM sodium chloride; FIG. 5-H is a graph of statistics of root length of 7-day-old Arabidopsis plants treated for 5 days on MS medium containing 150mM sodium chloride; FIG. 5-I is the germination of Arabidopsis seeds grown for 6 days on MS medium with 75mM sodium chloride; FIG. 5-J are statistical plots of germination rates of Arabidopsis seeds grown for 6 days on MS medium with 75mM sodium chloride; FIG. 5-K is the germination of Arabidopsis seeds grown for 6 days on MS medium with 100mM sodium chloride; FIG. 5-L is a graph showing statistics of germination rates of Arabidopsis seeds grown for 6 days on MS medium containing 100mM sodium chloride; FIG. 5-M is a graph showing chlorophyll extraction results of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days; FIG. 5-N is a graph showing chlorophyll determination results of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days; FIG. 5-O is a graph showing the results of conductivity measurement of 20-day-old Arabidopsis plants after treatment with 200mM sodium chloride for 15 days. The measurement of the indexes is an important measurement index for evaluating the salt resistance of the transgenic plant, and as can be seen from FIG. 5, the gene PbVHA-B1 enhances the salt resistance of the transgenic plant.

2. Histochemical stain analysis H₂O₂And O^2-Accumulation of

In transgenic lines (#1, #5, #6), lower conductivity indicates that they may have a greater ability to resist ROS than WT, and identification of the amount of ROS accumulated in the plant is necessary. Staining of plant leaves with DAB and NBT histochemical staining for detection of hydrogen peroxide (H)₂O₂) And superoxide anion (O)₂ ^-) The content of (D) was determined in comparison with the Wild Type (WT) Arabidopsis strain.

The results are shown in FIG. 6. FIG. 6 shows the histochemical staining analysis H of the gene encoding PbVHA-B1 in Arabidopsis thaliana₂O₂And O₂ ^-Accumulation, FIGS. 6-A and 6-B show that 20-day-old Arabidopsis plants after 7 days of 200mM sodium chloride treatment were stained for H by reactive oxygen histochemical staining of untransformed plants and three transgenic lines with Nitro-Tetrazol (NBT) and Diaminobenzidine (DAB), respectively₂O₂(FIG. 6-A) and O₂ ^-(FIG. 6-B) dyeing; FIGS. 6-C and 6-D are the treatment of 7-day-old Arabidopsis plants on MS minimal medium containing 100mM sodium chloride for 3 days, and then the untransformed plants and three transgenic lines were treated with Nitro-tetrazole (NBT) and Diaminobenzidine (DAB), respectively, on H₂O₂(FIG. 6-C) and O₂ ^-(FIG. 6-D) staining was performed. As shown in FIGS. 6-A and 6-B, in the case of the leaves stained with DAB after 7 days of salt stress, the leaves of the wild type strain appeared brown in color with a significantly larger area and darker color than those of the transgenic strain, and in the case of the leaves stained with NBT, the wild type strain appeared blue in color and larger area than those of the transgenic strain; FIGS. 6-C and 6-D Arabidopsis tissue culture seedlings 7 days old, after 3 days of stress, DAB and DBT staining areas and color depths of wild type plants were significantly greater than those of transgenic line plants, indicating that the transgenic line plants had lower ROS (H) under salt stress than wild type plants₂O₂And O₂ ^-) And (4) accumulating.

3. Na in plant cells⁺And K⁺Determination of content

When the plants are subjected to salt stress, the osmotic potential difference easily causes the water loss of cells. Na for making cell absorb water normally from high salt environment, reducing cell osmotic potential and maintaining ion balance in cell⁺And carrying out reverse concentration gradient transportation, and discharging or partitioning the solution into vacuoles. In addition to this, K⁺Compartmentalization into vacuoles to produce K in the cytoplasm⁺Reduced signal, thereby activating high affinity K on plasma membrane⁺The transporter of (1). Activated high affinity K⁺The transporter can be specifically selected from low K⁺Environment will be more K⁺Transport into cells, thereby maintaining stable Na in cells⁺/H⁺And water stress is avoided. Thus in transgenic lines, Na⁺Lower content of K⁺Higher levels indicate that they may have greater salt tolerance than WT.

Na⁺And K⁺The content determination steps are as follows: removing underground parts of the treated wild plants and transgenic plants, and placing the above-ground parts of the wild plants and transgenic plants in an oven at 105 ℃ for deactivation of enzymes of all tissue parts for 30 minutes to prevent ions from moving. And (3) after the water-removing, drying the sample in a 65 ℃ drying oven to constant weight, and generally drying for 3-4 days. Grinding the dried sample into powder, weighing 0.05g of the sample in a 15ml centrifuge tube, adding 2ml of 0.5mol/l HCl solution for leaching for 3 days, sucking out 1ml of HCl solution, adding 5ml of deionized water for leaching for one day and nightAfter the end, 1ml of leaching liquor is sucked and added into deionized water to be regulated to 10ml for standby. Preparation of Na⁺、K⁺Standard samples are used for preparing a standard curve, the used reagents are NaCl and KCl solutions, and the concentration of the standard samples is prepared between 0 and 100 mu g/ml according to requirements. Mixing the above leaching solution with Na⁺、K⁺The standard sample is measured by a flame photometer. Na in the sample⁺Or K⁺The content (. mu.g/g) was calculated according to formula I.

Na⁺Or K⁺Content (μ g/g) ═ ρ × dilution times total volume/dry weight of sample taken.

The results are shown in FIG. 7, and FIG. 7 shows the genes encoding PbVHA-B1, Arabidopsis thaliana and wild type Arabidopsis thaliana Na⁺And K⁺And (4) content. FIGS. 7-A and 7-B are wild type plants and three transgenic lines Na after treatment of 20-day-old Arabidopsis plants with 200mM sodium chloride solution for 15 days⁺And K⁺Content, FIG. 7-C is Na⁺And K⁺The ratio of the contents. FIG. 7-A Na in transgenic line⁺The content is 1/2 of wild type; k in FIG. 7-B⁺The content is higher and is 2 times of that of the wild type; wild type Na in FIG. 7-C⁺/K⁺The content is 3-4 times of that of the transgenic plants. The result shows that the over-expression of PbVHA-B1 can better promote K pairing of plants⁺And supply Na⁺The driving force required by vacuole regionalization maintains the stability of cell osmotic potential, thereby improving the salt tolerance of plants.

Example 6

PbVHA-B1 virus silencing plant resistance identification

1. Salt resistance assay of virus-silenced birchleaf pear seedlings

In order to identify whether the gene coded by PbVHA-B1 is closely related to plant salt tolerance, wild type and virus-silenced positive birch seedlings are subjected to salt stress for a certain time. Irrigating the well-grown wild-type and virus-silenced positive birch pear seedlings (OE1, OE2 and OE3) with 200mM sodium chloride solution under the same culture condition for 20 days, observing the treated phenotype, and measuring conductivity, chlorophyll, etc

FIG. 8 is a phenotype and physiological index assay of gene silencing lines encoding PbVHA-B1 and Wild Type (WT) sodium chloride treatment before and after treatment, wherein FIG. 8-A is the phenotype of 45-day-old birch pear seedlings before 20 days of 200mM sodium chloride treatment; FIG. 8-B is the phenotype of 45-day-old seedlings of Pyrus betulaefolia after 20 days of 200mM NaCl treatment; FIG. 8-C is the results of conductivity measurements of 45-day-old seedlings of Pyrus betulaefolia after 20 days of treatment with 200mM salt solution; FIG. 8-D is chlorophyll extraction from 45-day old seedling of Pyrus betulaefolia after 20 days of treatment with 200mM salt solution; FIG. 8-E is a chlorophyll determination of 45-day old seedlings of Pyrus betulaefolia after 20 days of treatment with 200mM salt solution; FIG. 8-F is a photograph of fluorescent chlorophyll from 45-day-old seedlings of Pyrus betulaefolia taken 20 days before treatment with 200mM sodium chloride; FIG. 8-G is the phenotype of 45-day-old seedlings of Pyrus betulaefolia after 20 days of 200mM NaCl treatment. The results in FIG. 8 show that silencing of the gene encoding PbVHA-B1 reduced salt tolerance in seedlings of Pyrus betulaefolia, and that the conductivity of positive seedlings was significantly increased compared to Wild Type (WT) and chlorophyll content was significantly decreased compared to wild type.

2. Histochemical stain analysis H₂O₂And O^2-Accumulation of

Staining of plant leaves with DAB and NBT histochemical staining method and detection of accumulated hydrogen peroxide (H) in plants₂O₂) And superoxide anion (O)₂ ^-) The content of (a).

The results are shown in FIG. 9. FIG. 9 is chemical staining analysis H of tissue of leaf of PbVHA-B1-encoded, gene-silenced Pyrus pyrifolia seedlings₂O₂And O₂ ^-Accumulation, FIGS. 9-A and 9-B show the active oxygen histochemical staining of wild type and three silent lines (DAB) for H respectively using Nitrotetrazolium (NBT) and Diaminobenzidine (DAB) after 7 days of 200mM NaCl treatment of 45-day-old leaves of birch pear seedlings₂O₂(FIG. 9-A) and O₂ ^-(FIG. 9-B) staining was performed. In contrast to transgenic Arabidopsis, as shown in FIGS. 9-A and 9-B, in which leaves stained with DAB and NBT after 7 days of salt stress, the leaf area and color depth of the virus silencing line were significantly larger than those of wild type Pyrus pyrifolia seedlings, indicating that silencing of the gene encoded by PbVHA-B1 leads to ROS (H) in plant cells₂O₂And O₂ ^-) Excessive accumulation of the plant cell membrane, more serious damage to the cell membrane and salt tolerance of the plantAnd (5) reducing.

3. Comprehensive analysis shows that the function of the PbVHA-B1 gene is identified after the gene is transferred into Arabidopsis, and the salt resistance of a transgenic over-expression strain is greatly improved compared with that of a control wild type. When salt stress occurs, the growth state of the transgenic arabidopsis is better than that of the wild arabidopsis through the measurement of indicators such as chlorophyll content, electric conductivity, germination rate, root growth condition and the like. Simultaneous transformation of hydrogen peroxide (H) in plants₂O₂) And superoxide anion (O)₂ ^-) The content activity of the compound is lower than that of a wild type, the active oxygen residue in a plant body is lower, and the cell damage is smaller. In transgenic lines, lower Na⁺Content and higher K⁺The content shows that the transgenic strain can better transport transmembrane ions, maintain the balance of intracellular osmotic potential and prevent the toxicity caused by water stress and ion accumulation. Meanwhile, after the PbVHA-B1 gene in the birch pear seedlings is silenced, salt resistance of the wild birch pear seedlings is superior to that of the silenced birch pear seedlings by observing phenotypes of the wild birch pear seedlings and the gene-silenced birch pear seedlings after salt stress, measuring chlorophyll content and conductivity of the wild birch pear seedlings and measuring ROS staining, and the silencing of the PbVHA-B1 gene causes the salt resistance of the birch pear seedlings to be weakened.

Analysis of the results shows that the PbVHA-B1 gene is closely related to salt tolerance, and the over-expressed PbVHA-B1 gene can effectively enhance the active oxygen scavenging capacity of transgenic plants, enhance the V-ATPase activity to provide energy required for transmembrane ion transportation, maintain the intracellular ion balance and osmotic potential steady state, and further improve the salt tolerance of the plants.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing university of agriculture

<120> pyrus betulaefolia vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement

<160>14

<170>SIPOSequenceListing 1.0

<210>1

<211>490

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Met Ala Val Ser Gln Asn Asn His Asp Met Asp Glu Gly Asn Leu Glu

1 5 10 15

Val Gly Met Glu Tyr Arg Thr Val Ser Gly Val Ala Gly Pro Leu Val

20 25 30

Ile Leu Glu Lys Val Lys Gly Pro Lys Phe Gln Glu Ile Val Asn Ile

35 40 45

Arg Leu Gly Asp Gly Thr Thr Arg Arg Gly Gln Val Leu Glu Val Asp

50 55 60

Gly Glu Lys Ala Ile Val Gln Val Phe Glu Gly Thr Ser Gly Ile Asp

65 70 75 80

Asn Lys Tyr Thr Thr Val Gln Phe Thr Gly Glu Val Leu Lys Thr Pro

85 90 95

Val Ser Leu Asp Met Leu Gly Arg Ile Phe Asn Gly Ser Gly Lys Pro

100 105 110

Ile Asp Asn Gly Pro Pro Ile Leu Pro Glu Ala Tyr Leu Asp Ile Ser

115 120 125

Gly Ser Ser Ile Asn Pro Ser Glu Arg Thr Tyr Pro Glu Glu Met Ile

130 135 140

Gln Thr Gly Ile Ser Thr Ile Asp Val Met Asn Ser Ile Ala Arg Gly

145 150 155 160

Gln Lys Ile Pro Leu Phe Ser Ala Ala Gly Leu Pro His Asn Glu Ile

165 170 175

Ala Ala Gln Ile Cys Arg Gln Ala Gly Leu Val Lys Arg Leu Glu Lys

180 185 190

Ser Glu Ser Leu Leu Asp Ala Gly Asp Val Glu Asp Asp Asn Phe Ala

195 200 205

Ile Val Phe Ala Ala Met Gly Val Asn Met Glu Thr Ala Gln Phe Phe

210 215 220

Lys Arg Asp Phe Glu Glu Asn Gly Ser Met Glu Arg Val Thr Leu Phe

225 230 235 240

Leu Asn Leu Ala Asn Asp Pro Thr Ile Glu Arg Ile Ile Thr Pro Arg

245 250 255

Ile Ala Leu Thr Thr Ala Glu Tyr Leu Ala Tyr Glu Cys Gly Lys His

260 265 270

Val Leu Val Ile Leu Thr Asp Met Ser Ser Tyr Ala Asp Ala Leu Arg

275 280 285

Glu Val Ser Ala Ala Arg Glu Glu Val Pro Gly Arg Arg Gly Tyr Pro

290 295 300

Gly Tyr Met Tyr Thr Asp Leu Ala Gln Ile Tyr Glu Arg Ala Gly Arg

305 310 315 320

Ile Glu Gly Arg Lys Gly Ser Ile Thr Gln Ile Pro Ile Leu Thr Met

325 330 335

Pro Asn Asp Asp Ile Thr His Pro Thr Pro Asp Leu Thr Gly Tyr Ile

340 345 350

Thr Glu Gly Gln Ile Tyr Ile Asp Arg Gln Leu His Asn Arg Gln Ile

355 360 365

Tyr Pro Pro Ile Asn Val Leu Pro Ser Leu Ser Arg Leu Met Lys Ser

370 375 380

Ala Ile Gly Glu Gly Met Thr Arg Arg Asp His Ser Asp Val Ser Asn

385 390 395 400

Gln Leu Tyr Ala Asn Tyr Ala Ile Gly Lys Asp Val Gln Ala Met Lys

405 410 415

Ala Val Val Gly Glu Glu Ala Leu Ser Ser Glu Asp Leu Leu Tyr Leu

420 425 430

Glu Phe Leu Asp Lys Phe Glu Lys Lys Phe Val Ser Gln Gly Ala Tyr

435 440 445

Asp Thr Arg Asn Ile Phe Gln Ser Leu Asp Leu Ala Trp Thr Leu Leu

450 455 460

Arg Ile Phe Pro Arg Glu Leu Leu His Arg Ile Pro Ala Lys Thr Leu

465 470 475 480

Asp Leu Phe Tyr Ser Arg Asp Ala Ala Asn

485 490

<210>2

<211>1473

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atggctgttt cacaaaacaa tcatgacatg gacgagggaa acctagaggt cggaatggaa 60

tacagaactg tgtctggtgt ggccggacct ctggttatcc ttgaaaaagt taagggacct 120

aagtttcaag agattgttaa cattcgtttg ggagatggaa caactcgacg tggtcaagtc 180

ctggaagttg atggagagaa agctattgta caggttttcg aaggaacatc tggaattgac 240

aacaagtaca ctactgtgca attcacagga gaggttttga aaactccagt ctcacttgac 300

atgcttgggc gcatctttaa tggctctggg aagcccattg ataatggccc ccctattttg 360

cctgaggctt acctagacat atctgggagt tctattaatc cgagtgagag aacatatcct 420

gaagaaatga ttcagactgg aatttctact attgatgtca tgaactccat tgcgagagga 480

caaaaaatcc cccttttctc tgctgctggt cttcctcata atgaaatagc tgctcagata 540

tgtcgccagg ccggtttggt caagcggttg gagaaatctg agagtcttct tgacgctggg 600

gacgtagaag acgacaactt tgccattgtg tttgcagcta tgggagtaaa tatggagact 660

gcacagttct ttaagcgtga ttttgaggaa aatggttcaa tggagagagt gacccttttt 720

ctgaatctgg caaatgaccc tacaattgaa cgcattatta ctcctcgtat tgctcttact 780

actgcagaat atttggcata tgaatgtggg aagcatgttc ttgtcattct cactgatatg 840

agttcttatg ctgatgctct tcgtgaggtg tctgctgccc gagaggaagt gccgggaagg 900

cgtggatacc ctgggtacat gtatactgat ctggcacaaa tctatgagcg tgctggaaga 960

attgaagggc gaaaaggctc tattacccaa attccgatct taactatgcc aaatgatgat 1020

attacccacc ccactccaga tcttactgga tatattactg agggacagat atacattgac 1080

aggcagctcc acaacagaca gatatatcca ccaatcaatg tcctcccatc actatctcgt 1140

ctgatgaaga gtgctattgg tgaaggcatg actcgccggg atcattctga tgtatcaaat 1200

cagttatatg caaattatgc tattgggaag gatgtccagg caatgaaagc tgtggtcgga 1260

gaagaagcac tttcttcgga ggacttgcta tacctggagt tcttggacaa atttgagaag 1320

aagtttgtgt cccaaggagc ctatgacacc cgtaacatct tccagtccct cgatttggca 1380

tggacgttgc tgcgaatctt cccccgtgag cttctccacc gtatacctgc aaagaccctt 1440

gacctgttct acagcagaga tgcagctaat tga 1473

<210>3

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atggctgttt cacaaaacaa tca 23

<210>4

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

tcaattagct gcatctctgc tgta 24

<210>5

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tcctcggatt ccattgccca gc 22

<210>6

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

cacaagaaca gtttccttgg ttttc 25

<210>7

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

tgggctttgc tcctcttac 19

<210>8

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ccttcgtgct catcttacc 19

<210>9

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gagaacacgg gggactctag aatggctgtt tcacaaaaca atca 44

<210>10

<211>45

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gcccttgctc accatggatc cattagctgc atctctgctg tagaa 45

<210>11

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

atggctgttt cacaaaacaa tca 23

<210>12

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cgtcgtcctt gaagaagatg 20

<210>13

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

aaggttaccg aattctctag aatggctgtt tcacaaaaca atca 44

<210>14

<211>45

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

ggcctcgaga cgcgtgagct ctcaattagc tgcatctctg ctgta 45

Claims (9)

1. A birch pear vacuole proton pump PbVHA-B1 is characterized in that the amino acid sequence is SEQ ID No. 1.

2. The coding gene of the birch pear vacuole proton pump PbVHA-B1 is characterized in that the nucleotide sequence is SEQ ID No. 2.

3. A primer pair for amplifying the coding gene of claim 2, wherein the nucleotide sequence of the upstream primer in the primer pair is SEQ ID No. 3; the nucleotide sequence of the primer pair downstream primer is SEQ ID No. 4.

4. A plant expression vector comprising the encoding gene of claim 2.

5. The plant expression vector of claim 4, wherein the base vector in the plant expression vector is pcmcia 1300;

the multiple cloning sites of the coding gene inserted into the PCMBIA1300 are XbaI and BamHI.

6. Use of the vacuolar proton pump PbVHA-B1 in pyrus betulaefolia as claimed in claim 1, the coding gene as claimed in claim 2, the primer pair as claimed in claim 3, or the plant expression vector as claimed in claim 4 or 5 for breeding salt-tolerant plants or for breeding transgenic salt-tolerant plants.

7. The use of claim 6, wherein the salt comprises Na⁺Or K⁺。

8. Use according to claim 6 or 7, wherein the salt is present in a concentration of not more than 1000 mMol/L.

9. The use of claim 6, wherein the plant comprises Arabidopsis thaliana or Pyrus pyrifolia.

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