CN112011553A

CN112011553A - Lipid transport protein and coding gene and application thereof

Info

Publication number: CN112011553A
Application number: CN202010875678.XA
Authority: CN
Inventors: 李楠楠; 李加纳; 肖忠春
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-12-01

Abstract

The invention belongs to the field of crop genetic breeding, and particularly relates to a lipid transfer protein, and a coding gene and application thereof. The gene encoding the lipid transporter comprises the amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 3. The gene encodes a lipid transporter comprising: as shown in SEQ ID NO: 2; or as shown in SEQ ID NO: 4; or said SEQ ID NO: 2 by substitution and/or deletion and/or addition of one or more amino acid residues; or said SEQ ID NO: 4 by substitution and/or deletion and/or addition of one or more amino acid residues. The lipid transfer protein and the coding gene thereof provided by the invention can obviously improve the biomass of plants and the oil content of grains.

Description

Lipid transport protein and coding gene and application thereof

Technical Field

The invention belongs to the field of crop genetic breeding, and particularly relates to a lipid transfer protein, and a coding gene and application thereof.

Background

In plants, de novo fatty acid (Fas) synthesis occurs in the chloroplast stroma with acyl chain growth attached to an Acyl Carrier Protein (ACP) and is used for lipid assembly mainly in the form of C16: 0-and C18:1-ACP (Li-Beisson et al 2013; Troncoso-Ponce et al 2016). Although it is widely believed that free FAs is transported across the plastid membrane, its transport mechanism is not clear, and recent studies have found that arabidopsis FAX1 is a novel membrane protein localized on the chloroplast inner membrane involved in fatty acid transport (Li et al 2015). Analysis of the arabidopsis FAX1 deletion mutant and overexpression mutant shows that the arabidopsis FAX1 gene plays an important role in the processes of biological yield formation, pollen fertility and formation of fatty acid derivatives such as lipid, ketone wax and pollen cell wall composition substances. When the FAX1 gene was deleted, mass spectrometry analysis revealed that lipids on the endoplasmic reticulum decreased and lipids in the chloroplasts increased, the opposite was true when the FAX1 gene was overexpressed, and Triacylglycerols (TAGs) in flowers and leaves increased significantly. Compared with wild arabidopsis thaliana, the biological yield of the FAX1 gene deletion mutant is obviously reduced, and the biological yield of the overexpression mutant is obviously increased. In yeast cells, the AtFAX1 gene also mediates fatty acid transport. Thus, FAX1 plays a crucial role in the transport of fatty acids across the inner membrane of the chloroplast. And a recent study showed that the specific expression of the AtFAX1 gene in Arabidopsis seeds increases the oil content of the seeds (Tian et al.2018).

Through gene transfer and recombination, rape breeding is improved towards multiple targets, such as high quality, high yield, ultra-high yield, multiple resistance and the like, and outstanding achievements are obtained in genetic improvement of multiple target characters. With the increasing demand of people for vegetable oil and the higher requirement on quality, it is obvious that the conventional breeding method cannot meet the large demand. Therefore, the research is expected to improve the oil content of the brassica napus seeds by researching the function of a gene FAX1 which is reported to be positioned on the chloroplast inner membrane to participate in oil transfer in the formation of the oil content of the brassica napus in arabidopsis.

Disclosure of Invention

The invention aims to provide a gene for coding a lipid transporter, which comprises a nucleotide sequence shown as SEQ ID NO: 1 or SEQ ID NO: 3.

Further, the gene is used for identifying candidate genes related to the oil content by QTL and GWAS analysis of the oil content character and combining with transcriptome analysis of extremely high and low oil content materials. Meanwhile, the membrane protein BnaFAX family genes related to lipid transfer are analyzed, and finally, the main functional genes BnaFAX1-1 and BnaFAX1-2 with oil content are screened out.

Further, the nucleotide sequence of the gene BnaFAX1-1 is SEQ ID NO: 1, the nucleotide sequence of the BnaFAX1-2 is SEQ ID NO: 3, respectively.

Further, the gene comprises a nucleotide sequence which has no less than 90% of identity with SEQ No.1 or SEQ No. 3.

The present invention aims to provide a gene-encoded lipid transporter.

The lipid transporter comprises: as shown in SEQ ID NO: 2; or as shown in SEQ ID NO: 4; or said SEQ ID NO: 2 by substitution and/or deletion and/or addition of one or more amino acid residues; or said SEQ ID NO: 4 by substitution and/or deletion and/or addition of one or more amino acid residues.

Further, the lipid transporter protein belongs to a plant lipid transporter protein.

The present invention aims to provide a biomaterial of a gene encoding a lipid transporter.

The biological material is as follows: an expression cassette comprising said gene; or a recombinant vector comprising said gene/said expression cassette; or a recombinant plasmid comprising said gene/said expression cassette; or a recombinant bacterium comprising said gene/said expression cassette/said recombinant vector/said recombinant plasmid; or a transgenic plant cell line comprising said gene/said expression cassette/said recombinant vector.

The invention aims to provide an application of the plant in improving the biomass and/or the oil content of grains.

Further, the plant is rape.

The invention aims to provide a method for improving the biomass and/or the oil content of grains of a plant, wherein the method is used for introducing the gene into the plant.

Further, the gene is BnaFAX1-1 and/or BnaFAX 1-2.

Further, the plant is rape.

The invention aims to provide a method for constructing a transgenic plant with high plant biomass and/or high kernel oil content, and the method is characterized in that the lipid transporter is overexpressed in the plant.

The invention has the beneficial effects that:

the lipid transfer protein and the coding gene thereof provided by the invention can obviously improve the biomass of plants and the oil content of grains.

The transgenic method with high biomass and high oil content provided by the invention is convenient to operate, and is simple and feasible.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows the CDS sequence amplification of BnaFAX1-1(a) and BnaFAX1-2(b) according to the present invention.

FIG. 2 shows the 500mg/L Basta resistance assay of transgenic plants of the invention. Wherein (a) leaf of wild type control plant (b) leaf of anti-Basta positive plant.

FIG. 3 shows the expression pattern of the transgenic BnaFAX1-1 of the present invention in the tissues of Zhongshuang 11.

FIG. 4 shows the results of qRT-PCR analysis of gene expression levels of transgenic plants of the invention.

FIG. 5 is the statistics of Bnafax1-1 overexpression of Brassica napus in seedling stage (32d) phenotype (A) phenotype of whole plant (B) phenotype of leaf (C) phenotype. 10 individuals were selected for each strain for phenotypic determination.

FIG. 6 shows phenotype statistics of (A) a mature field BnaFAX1-1 overexpression transgenic plant phenotype, (B) a mature field BnaFAX1-1 overexpression transgenic plant single plant main inflorescence silique phenotype, (C) a mature field BnaFAX1-1 overexpression transgenic plant phenotype (including plant height, main inflorescence length, single plant effective branch number, main inflorescence silique number, single plant silique number, silique length, silique number per silique, thousand seed weight, single plant economic yield and overground biological yield), and 8-10 plants are determined for each plant line.

FIG. 7 shows phenotype statistics of (A) a mature field BnaFAX1-2 overexpression transgenic plant phenotype, (B) a mature field BnaFAX1-2 overexpression transgenic plant single plant main inflorescence silique phenotype, (C) a mature field BnaFAX1-2 overexpression transgenic plant phenotype (including plant height, main inflorescence length, single plant effective branch number, main inflorescence silique number, single plant silique number, silique length, silique number per silique, thousand seed weight, single plant economic yield and overground biological yield), and 8-10 plants are determined for each plant line.

FIG. 8 is a graph of the oil content and protein content analysis of mature seeds of BnaFAX1-1 overexpression line of the present invention (a) and the fatty acid content analysis of mature seeds of BnaFAX1-1 overexpression line (b).

FIG. 9 is a graph of the oil content and protein content analysis of mature seeds of BnaFAX1-2 overexpression line of the present invention (a) and the fatty acid content analysis of mature seeds of BnaFAX1-2 overexpression line (b). And indicate T-test P <0.05 and P <0.01, respectively, with significant and very significant differences between wild-type and transgenic lines, respectively.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1

RNA was extracted from young leaves of Brassica napus (Brassica napus) by using the crude EZ-10 DNAway RNA extraction kit, and its mRNA was reverse-transcribed into cDNA using the reverse transcription kit of Takara corporation (FIG. 2A); using the cDNA as PCR template, using easy Taq enzyme and BnaFAX1 gene specific primer BnaFAX1-1 FP (XbaI) 5'-TCTAGAATGGCGACGAAAATCTCTCACC-3' (shown as SEQ ID NO: 5 in the sequence table), BnaFAX1-1RP (SacI) 5'-GAGCTCTCAGTGTGAAGGGCTAGTAGATGG-3' (shown as SEQ ID NO: 6 in the sequence table), BnaFAX1-2 FP (XbaI) 5'-TCTAGAATGGCGACGAAAATCTCTCAC-3' (shown as SEQ ID NO: 7 in the sequence table), BnaFAX1-2RP (SacI) 5'-GAGCTCTCAGTGTGAAGGGCTAGTAGATG-3' (shown as SEQ ID NO: 8 in the sequence table) to amplify BnaFAX1-1 and BnaFAX1-2 fragments, the PCR method and program are as follows:

TABLE 1PCR methods and procedures

Composition of	Volume of
		10mMdNTPs Mixture	1μl
10uM Forward Primer	1μl
		10uM Reverse Primer	1μl
Template	2μl
		10x Easy Taq buffer(Mg+)	5μl
EasyTaq enzyme (5U/uL)	0.5μl
		ddH2O	39.5μl
Total	50μl

The specific PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 2min → (94 ℃ 45s → 56 ℃ 45s → 72 ℃ 1 min.) the process proceeds through 35 amplification cycles → extension at 72 ℃ for 10min → storage at 4 ℃. The PCR product was checked for quality by electrophoresis on a 1% agarose gel. A band of about 680bp was detected by 1% agarose gel electrophoresis and matched with the band of interest (FIG. 1). And recovering the target fragment, connecting the target fragment with a pMD19-T vector, transforming escherichia coli, detecting bacterial liquid, selecting several bacterial liquids with correct bacterial liquid detection, and sending the bacterial liquids to a sample for sequencing, wherein the obtained sequence is consistent with a reference sequence.

The T vector connection is specifically as follows:

the following reagents were mixed in a 500uL sterile Eppendorf tube on an ice-water bath:

TABLE 2 reagent Components

After gently mixing with a pipette tip, the mixture was ligated overnight at 16 ℃.

Example 2

Transforming the extracted recombinant plasmid into agrobacterium tumefaciens competent cells, and the specific operation steps are as follows:

(1) taking out the agrobacterium strain competent cell GV3101 from a refrigerator at the temperature of-80 ℃, unfreezing the agrobacterium strain competent cell GV3101 on ice, then slightly adding 5ul of over-expression engineering strain plasmid along the tube wall, and slightly and uniformly mixing the over-expression engineering strain plasmid with a gun head;

(2) ice water bath for 5min, rapidly cooling in liquid nitrogen for 8min, and heat shock in 37 deg.C water bath for 5 min;

(3) adding 900ul of YEB (adding 25mg/L of streptomycin Str and 20mg/L of rifampicin Rif) at room temperature, and performing shake culture at 28 ℃ and 180-200 rpm for 3-5 h for resuscitation;

(4) after resuspension, centrifuge the tube at 5,000rpm for 5min at room temperature, aspirate the supernatant (800 ul), and resuspend the cells, and spread evenly on LB solid plates (adding antibiotics Str 25mg/L, Rif 20mg/L, and Kan 50 mg/L);

(5) placing the plate in an incubator at 28 ℃ for 2d in an inverted way until a monoclonal colony with a proper size grows on the plate, picking the monoclonal colony in a sterilized 10ul tip in 900ul YEB liquid medium (added with antibiotics Str 25mg/L, Rif 20mg/L and Kan 50mg/L), and culturing the monoclonal colony in a shaker at 28 ℃ and 250rpm for 2 d;

(6) carrying out PCR detection on the bacteria liquid shaken to a proper concentration, selecting clones with positive PCR detection, carrying out sample-feeding sequencing, selecting two agrobacterium bacteria liquids with correct sequencing, adding 50% of glycerol to a final concentration of about 15%, carrying out sample-feeding to Wuhan double helix company to transform the Brassica napus Westar variety, or storing in a refrigerator at the temperature of-80 ℃ for later use.

Example 3

(1) Transgenic plant Basta detection

Uniformly smearing 200mg/L Basta solution on the surface of the regenerated plant leaf, observing the change condition of the leaf (the yellowing of the leaf is negative, and the normal growth is positive) after 3-5 days, and preliminarily judging the Basta resistance of the regenerated plant (figure 2).

(2) PCR detection of transgenic plants

The total genome DNA of the transgenic regeneration plant is extracted according to the DNA extraction method, and the qualified genome DNA is used for PCR identification of transgene insertion after electrophoresis detection and spectrophotometer detection. The primer combination F35S3ND (shown as SEQ ID NO: 15 in the sequence table) + gene rear primer, gene front primer + NOS5ND (shown as SEQ ID NO: 16 in the sequence table) and FBar (shown as SEQ ID NO: 17 in the sequence table) + RBar (shown as SEQ ID NO: 18 in the sequence table) are used for detecting the regenerated plants over-expressed by BnaFAX1-1 and BnaFAX 1-2. The PCR reaction system and the gene amplification program are consistent with the gene amplification.

Example 4

The specific operation method of the real-time fluorescent quantitative PCR (qRT-PCR) method is carried out according to the instruction of a TaRaKa TB Green Premix Ex Taq II quantitative kit, and reference gene primers: BnActin 7-FP: 5'-TGGGTTTGCTGGTGACGAT-3' (shown as SEQ ID NO: 9 in the sequence Listing); BnActin 7-RP: 5'-TGCCTAGGACGACCAACAATACT-3' (shown as SEQ ID NO: 10 in the sequence list), target gene primer: qRT-BnaFAX1-1 FP: 5'-GATGGGAACAGCTCAGAAACAC-3' (shown as SEQ ID NO: 11 in the sequence Listing); qRT-BnaFAX1-1 RP: 5'-GTTCCTCTACTGTCTCAGTGA-3' (shown as SEQ ID NO: 12 in the sequence Listing); qRT-BnaFAX1-2 FP: 5'-CCTTAGGTATCGCCACTTGTCT-3' (shown as SEQ ID NO: 13 in the sequence Listing); qRT-BnaFAX1-2 RP: 5'-CTCGGTGATAATCTCCTTAAT-3' (shown as SEQ ID NO: 14 in the sequence Listing). Firstly, preparing in a 96-hole quantitative PCR plate according to the following reaction system:

TABLE 3 reaction System

Followed by a CFX96 Touch^TMThe following program was run on a fluorescent quantitative PCR instrument (Bio-Rad): at 95 ℃ for 3 min; 95 ℃ for 10 s; 30s at 60 ℃; 40 cycles, 65 ℃ for 5 s. The annealing temperature varied depending on the primer, and BnACT7(Chen et al.2010c) was used as an internal reference gene in this study. The CFX-Manager software from Bio-Rad was used for data collection and subsequent processing analysis.

After the program is finished, the product unicity and the dissolution temperature are judged by using the Tm value of the dissolution curve. And obtaining the relative expression quantity of the target gene relative to the internal reference gene through the numerical value of the ratio of the target gene to the reference gene, and finally comparing the relative expression quantity among the samples. The data results in fig. 3 show that: bnafax1 was expressed in the highest amount in leaves and also in higher amounts in siliques at different developmental stages.

Example 5 detection of expression level of Bnafax1 Gene in wild type rape and transgenic rape

Taking the leaves of the same-period wild rape and the transgenic rape with good growth state, extracting RNA, synthesizing cDNA by reverse transcription, and carrying out the detection experiment of real-time fluorescence quantitative PCR by using the cDNA as a template (the method is the same as the above). The results of overexpression of the expression level of BnaFAX1 gene in oilseed rape by wild type and different lines are shown in FIG. 4.

Wherein (a) the qRT-PCR comparison of the BnaFAX1-1 gene expression level in the BnaFAX1-1 transgenic line leaves and the control plants (b) the qRT-PCR comparison of the BnaFAX1-2 gene expression level in the BnaFAX1-2 transgenic line leaves and the control plants. BnActin7 was used as an internal reference gene. Westar served as wild type control. WT: a wild type; OE #17, OE #19, OE #20 and OE #21 are overexpression transgenic lines of BnaFAX1-1 in Brassica napus; OE #26, OE #27, OE #29 and OE #30 are overexpression transgenic lines of BnaFAX1-2 in Brassica napus.

Compared with wild BnaFAX1 gene, the expression level of the BnaFAX1-1 and BnaFAX1-2 transgenic plants is respectively improved by 6.79-85.83 and 48.44-127.17 times.

Example 6 functional verification of BnafAX1 Gene increasing oilseed rape Biomass in wild type oilseed rape and transgenic oilseed rape

To test the seedling growth of BnaFAX1-1 overexpressing Brassica napus, three overexpressing lines (i.e., OE #17, OE #19, and OE #21) were selected for nutrient culture in an incubator, with untransformed Westar wild type Brassica napus as a control (WT), and phenotypical observations and measurements were made when the oilseed rape was grown up to 32 days. The results show that BnaFAX1-1 over-expressed Brassica napus grows significantly more vigorously at the seedling stage than the control plants (FIG. 5A), and the leaves are also larger than the control plants (FIG. 5B). The root system is analyzed by a Wanshen LA-S series plant image analyzer, and the result shows that the total root length, the root area, the root surface area, the root volume, the root fresh weight and the root dry weight of a transgenic plant line are obviously higher than those of a control except that the main root length is not obviously different from that of the control. In addition, the fresh leaf weight, dry leaf weight, leaf length, leaf width and leaf area of the transgenic lines were significantly higher than the control (fig. 5C). These results show that the expression of BnaFAX1-1 gene in Brassica napus is promoted, and the growth and development of plants can be effectively promoted.

After the seeds are mature, the phenotypes of 4 BnaFAX1-1 overexpression transgenic cabbage type rape strains and control plants are observed and measured, and investigation statistics show that the plant heights of the transgenic plants and a Westar control (referred to as a control) are remarkably different, the transgenic strains except OE #17 show the result, and the effective branch number of the transgenic strains is remarkably larger than that of the control (FIGS. 6A and C). Although there was no significant difference in the length of the main inflorescence between the transgenic plants and the Westar control, the number of main inflorescence siliques of each transgenic line was significantly more than the control (FIG. 6B, C). Compared with the control, although the thousand kernel weight of the seeds of each transgenic line is not obviously different, the single plant silique number, the silique length and the kernel number of each transgenic line are obviously increased, and finally the single plant economic yield of each transgenic line is obviously improved. In addition, the total overground biomass yield dry weight was measured by weighing, and the results showed that the overground biomass yield of each transgenic line was significantly higher than that of the control (fig. 6C). These results further indicate that promoting the expression of BnaFAX1-1 gene in Brassica napus can effectively promote the growth and development of plants and effectively increase the economic yield of Brassica napus.

The results of the determination of BnaFAX1-2 over-expressing Brassica napus are substantially consistent with the results of the transgene over-expressing BnaFAX1-1 (FIG. 7).

Example 7 functional verification of Bnafax1 Gene affecting seed oil and fatty acid content in wild-type and transgenic oilseed rape

By measuring the oil content and protein content in the mature seeds of the over-expression lines of 4 BnaFAX1-1 Brassica napus, the results are shown in FIG. 8a, the oil content in the mature seeds of each transgenic line is significantly higher than that of the control, and the protein content is not significantly different. Further analysis of the fatty acid content of mature seeds showed (FIG. 8b) that the oleic acid (C18:1) content was significantly higher in the seeds of each transgenic line, while the eicosanoid (C20:0) and erucic acid (C22:1) content was significantly lower in the seeds of each transgenic line compared to the wild-type control seeds. The content of linoleic acid (C18:2) showed a significant reduction in all three transgenic lines (OE #17, OE #19 and OE #21) compared to wild type control seeds, whereas the content of linolenic acid (C18:3) showed a very significant reduction only in OE #17, whereas the difference was not significant in the other three transgenic lines. Also, the results were consistent in BnafAX1-2 overexpressing Brassica napus (FIG. 9).

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Sequence listing

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Claims

1. A gene encoding a lipid transporter protein, wherein the gene comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

2. The gene of claim 1, wherein said gene comprises a nucleotide sequence having no less than 90% identity to SEQ No.1 or said SEQ No. 3.

3. The gene-encoded lipid transporter protein of claim 1 or 2, wherein the lipid transporter protein comprises: as shown in SEQ ID NO: 2; or as shown in SEQ ID NO: 4; or said SEQ ID NO: 2 by substitution and/or deletion and/or addition of one or more amino acid residues; or said SEQ ID NO: 4 by substitution and/or deletion and/or addition of one or more amino acid residues.

4. The lipid transporter according to claim 3, wherein the lipid transporter belongs to the plant lipid transporter.

5. Biomaterial comprising the genes of claim 1 or 2, characterized in that it is: an expression cassette comprising said gene; or a recombinant vector comprising said gene/said expression cassette; or a recombinant plasmid comprising said gene/said expression cassette; or a recombinant bacterium comprising said gene/said expression cassette/said recombinant vector/said recombinant plasmid; or a transgenic plant cell line comprising said gene/said expression cassette/said recombinant vector.

6. Use of the gene of claim 1 or the lipid transport protein of claim 3 or the biological material of claim 5 for increasing plant biomass and/or kernel oil content.

7. Use according to claim 6, wherein the plant is oilseed rape.

8. Method for increasing the oil content in biomass and/or grain of a plant, characterized in that a gene according to claim 1 or 2 is introduced into said plant.

9. The method of claim 8, wherein the plant is canola.

10. Method for constructing a transgenic plant with increased biomass and/or kernel oil content in plants, characterized in that a lipid transporter protein according to claim 3 or 4 is overexpressed in the plant.