CN111978386B - Rice chloroplast protein OsFBN7 and application thereof - Google Patents

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a rice chloroplast protein OsFBN7 and a function thereof in regulating and controlling a head-to-head synthesis path of fatty acid in rice chloroplasts. The invention discovers that the coding gene of chloroplast protein OsFBN7 is constitutively overexpressed in rice, and the obtained transgenic plant shows double phenotypes of obviously improving the contents of diglyceride and glycolipid in leaves and the contents of triglyceride and free fatty acid in mature seeds compared with a wild plant. The OsFBN7 protein and the coding gene thereof have important application prospects in the cultivation and identification of high-fat plant varieties.

Description

Rice chloroplast protein OsFBN7 and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a rice chloroplast protein OsFBN7 and application thereof.

Background

The lipid is not only a high-efficiency energy storage substance in a living body, but also a main component and an important signal molecule of a biological membrane, is widely involved in various life activity processes, and is an essential component required for plant growth and development. Fatty acids are essential precursors in the synthesis of glycolipids, phospholipids and storage lipids, and the synthesis of the glycolipids and the phospholipids is hindered, so that the growth and development of plants are seriously influenced. Rice is one of the important food crops in the world, and when the lipid content of rice cells is insufficient or the synthesis of the rice cells is hindered, the rice cells directly influence the growth and development of the rice, possibly resulting in yield reduction and quality reduction.

Removing rice hulls from fatty acid paddy in the seeds to obtain the brown rice. The brown rice is composed of rice bran layer, embryo and endosperm. The testa oryzae layer accounts for about 10% of the total weight of the brown rice. The rice bran layer comprises pericarp, seed coat, endosperm and aleurone layer. Besides being rich in starch and protein, the brown rice also contains 2-5% of lipid. The oil and fat in brown rice are mainly distributed in the aleurone layer of the embryo and the rice bran layer. The main components of lipids are: triglyceride (TAG) 76.4-80.5%, Free Fatty Acids (FFAs) 7.2-9.8%, and Phospholipids (PLs) 3.5-3.6%. The main components of the free fatty acid are: 15-20% of palmitic acid (C16:0), 36-48% of oleic acid (C18:1) and 30-38% of linoleic acid (C18: 2). They can reduce cholesterol content, and treat concussion, hyperlipidemia, and cardiovascular and cerebrovascular diseases. Rice bran oil is a well-recognized nutritional and healthy oil at home and abroad (Birla et al, 2017; Yin et al, 2014; Qiu et al, 2008). The method has resource advantages in main rice production areas such as the middle and lower reaches of Yangtze river and the northeast rice area in China, and is a main rice bran oil processing area in China. However, the annual output of rice bran oil in China is less than 12 ten thousand tons, the utilization rate is less than 10 percent, and the development prospect and the potential for developing rice bran oil products are very large. Therefore, the development of high oleic acid rice varieties and production technologies and the development of nutritional and healthy rice have important social and economic benefits for the construction of the healthy Chinese 2030 programming compendium.

Chloroplast protein FBN chloroplasts are organelles characteristic of plant cells. It is composed of chloroplast two-layer envelope, thylakoid body and stroma. Thylakoid membranes and chloroplast inner membranes contain mainly monogalactosyl-diacylglycerol (MGDG), digalactosyl-diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) and small amounts of phospholipids. MGDG and DGDG account for 52% and 26% of chloroplast lipids, respectively (Kalisch et al, 2016).

In 1994, the carotenoid-bound 32kDa fibrillar protein, termed fibrin (chloroplast, FBN), was first isolated from the fibrous chromoplast fibrils of the pericarp of sweet pepper (Deruere, 1994). Later, FBN homologous proteins were isolated in plastid globules of potato leaves, pea leaves, arabidopsis leaves, cucumber corolla and rape anthers. Finally, these proteins derived from plastid globules are collectively referred to as Fibrin (FBN). Of the 14 AtFBNs in Arabidopsis, FBN1a/1b, FBN2, FBN4, FBN7a/7b and FBN8 are the core proteins of the plastid globule. FBN1a interacts with FBN1b, FBN1a/1b can interact with starch synthase SS4, and the biosynthesis of starch in chloroplast is affected (G a mez-Arjona et al,2014 a; G a mez-Arjona et al,2014 b). FBN4 affected the lipophilicity of the plastid globule in chloroplasts, chloroplast development and photooxidation resistance (Singh et al, 2010). Among 11 OsFBNs in rice, FBN5 was associated with photo-oxidative inhibition of rice (Otsubo et al, 2018; Kim et al, 2015); the overexpression of the FBN1 gene increases the number of plastid globules in chloroplasts and negatively regulates the heat resistance of rice in the flowering period (Li et al, 2019). However, there has been no report on the function of OsFBN7 protein so far.

Biosynthesis of fatty acids from head to head, the pathway of synthesis of fatty acids from head to head in rice chloroplasts has not been studied in depth at present (Kim et al, 2015; Qu et al, 2008). It is generally accepted that de novo synthesis, elongation and desaturation of fatty acids in plant chloroplasts occur primarily in the chloroplasts. The basic process is roughly as follows: pyruvate produces acetyl CoA under the action of a pyruvate dehydrogenase system located on the chloroplast envelope. In the chloroplast stroma, acetyl CoA carboxylase (ACCase) catalyzes the production of malonyl CoA, and ACP-malonyl monoacyl transferase (MCMT) catalyzes the production of malonyl-ACP. Then, acetyl-CoA and malonyl-ACP undergo a condensation-reduction-dehydration-re-reduction cycle, and the 4 enzymes catalyzing this cycle are KAS III (β -ketoacyl-ACP synthase III), KAR (β -ketoacyl-ACP reductase), HAD (β -hydroxyacyl-ACP dehydratase) and EAR (β -enoyl-ACP reductase) in this order, to synthesize butyryl-ACP. Similarly, C16:0-ACP was synthesized by 6 cycles under the influence of KAS I (β -ketoacyl-ACP synthase I), KAR, HAD and EAR. Finally, C18:0-ACP was synthesized under the action of KAS II (β -ketoacyl-ACP synthase II), KAR, HAD and EAR. Among them, KAS (β -ketoacyl-ACP synthsase) is the most important key enzyme for determining the elongation of fatty acid carbon chain. In particular, AtKAS I and OsKAS I affect chloroplast development, seed maturation and fatty acid accumulation processes (Wu & Xue, 2010; Ding et al, 2015). Secondly, free C16 and C18 fatty acids are synthesized under the action of acyl-ACP thioesterase (Fat), terminating the elongation of the fatty acid carbon chain. Rice contains 25 Fat genes, FatA and FatB being the most important. Among them, FatA specifically catalyzes production of oleic acid from oleoyl ACP (C18:1), and FatB catalyzes production of palmitic acid and stearic acid from stearoyl ACP and ACP, respectively (Salas et al, 2002). Fatty acid desaturases fall into two broad classes. The following: fatty acyl ACP desaturase. Rice has 7 Stearoyl ACP Desaturase (SAD) genes, all encoding chloroplast proteins. SAD2 and SAD5 are of the most importance. II, class II: free Fatty Acid Desaturase (FAD). Rice contains 20 genes: 4 OsFAD2 subfamily genes, 2 OsFAD3 subfamily genes, 1 OsFAD6, 1 OsFAD7, 1 OsFAD8, 9 OsFAB2 subfamily genes, 1 OsDES1 and 1 OsSLD 1. Among them, OsFAD2 and OsFAD3 are present in the endoplasmic reticulum, and OsFAD6, OsFAD7 and OsFAD8 are present in the chloroplast (E et al, 2019). An OsFAD2-1 gene knockout expression mutant is obtained by RNAi interference and CRISPR-Cas9 gene editing technology, oil drops in mature seeds are large, the content of oleic acid (C18:1) is obviously increased, and the content of linoleic acid (C18:2) is reduced (Abe et al, 2018; Tiwari et al, 2016; Zaplin et al, 2013).

Biosynthesis of MGDG and DGDGDG lysophosphatidic acid (LPA) is synthesized by reaction of C16:0-ACP or C18:1-ACP with glycerol-3-phosphate (G-3-P) catalyzed by phosphate-3-glycerol-acyltransferase (GPAT). Further synthesis of Phosphatidic Acid (PA) is carried out by LPA acyltransferase (LPAAT). Diacylglycerol (DAG) is then synthesized under the action of Phosphatidic Acid Phosphatase (PAP). In chloroplasts, both MGDG synthase (MGD) and DGDG synthase (DGD) are localized in the bilayer envelope of chloroplasts. MGD catalyzes DAG and guanosine diphosphate-galactose (UDP-Gal) to react to synthesize MGDG, and DGD catalyzes MGDG and UDP-Gal to react to synthesize DGDGDG. These glycolipids (MGDG, DGDG) are then mainly used for the synthesis and assembly of thylakoid membranes, chloroplast inner envelope and plastid globules.

Biosynthesis of oil bodies (TAGs) in the endoplasmic reticulum it is generally accepted that there is a pathway of synthesis-assembly-accumulation of oil bodies (TAGs) in the Endoplasmic Reticulum (ER) ((

&

2019). First, the substrates of the TAG synthesis pathway are derived from phospho-3-glycerol (G-3-P) and long-chain fatty acyl-CoA. In the chloroplast envelope, long-chain fatty acid CoA synthetase (LACS) catalyzes the synthesis of long-chain fatty acyl CoA, which is then transported to the ER. There are 5 LACS genes in rice. Thus, the introduction of long-chain fatty acyl-CoA into the ER determines the oil in the ER of rice cellsKey step in the synthesis of the body (TAG). Subsequently, under the catalysis of phosphate-3-glycerol-acyltransferase (GPAT), the long-chain fatty acyl CoA reacts with glycerol-3-phosphate to generate lysophosphatidic acid (LPA), and rice has 18 GPAT genes; further synthesis of Phosphatidic Acid (PA) is carried out by LPA acyltransferase (LPAAT). The rice has 2 LPAAT genes; diacylglycerol (DAG) is produced in the presence of Phosphatidic Acid Phosphatase (PAP). Rice has 3 PAP genes; triacylglycerols (TAGs) are synthesized under the action of DAG acyltransferase (DGAT). Rice has 2 DGAT genes. Among them, DGAT2 is important; in the ER, TAGs are packaged and stored in oil bodies (oil bodies) after their synthesis. The oil bodies consist of a monolayer of phospholipids and three classes of proteins, enveloped by a TAG core (Krahmer et al, 2009; Huang, 2018). Three lipid-binding protein genes of rice: 7 oleosin (Oleosins) genes, 9 calbindin (Caleosins) genes and 3 steroid binding protein (Steroleosins) genes (Murphy)&Vance,1999；Wu et al,2010)。

During rice seed maturation, de novo synthesized FAs in rice chloroplasts are converted to fatty acyl-CoA for translocation into the ER. In the ER, it reacts with glycerol-3-phosphate (G-3-P) to form TAG. TAG is the major lipid in the embryo and aleurone oil bodies, accounting for 76.4-80.5% of the total lipid content. Overexpression of 2 soybean Oleosin genes under the drive of an embryo-specific gene (REG-2) promoter increased oil content in rice seeds by 36.93% and 46.06%, respectively, but did not change the composition of fatty acids in TAG (Liu et al, 2012). Previous studies showed that EMS (ethyl methane sulfonate) chemically mutagenized rice mutant of OsKAS I gene has inhibited growth, reduced maturing rate, reduced total fatty acid content in seeds, and significantly reduced oleic acid (C18:1) (Ding et al, 2015). However, to date, there has been no report on the increase of fatty acid content in rice chloroplast and mature seed by OsFBN7 through positive regulation of OsKAS Ib activity.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a coding gene of rice chloroplast protein OsFBN7 in a way of regulating and controlling de novo synthesis of fatty acid in rice chloroplasts, and the coding gene is transferred into a target plant, so that the lipid and fatty acid contents in the chloroplasts and mature seeds can be improved, and the aim of cultivating the target plant with high oil content in the seeds is fulfilled.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a rice chloroplast protein OsFBN7, the amino acid sequence of which is shown in SEQ ID No.1, or the amino acid sequence which is formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.1 and has the same function, or the amino acid sequence which has at least 90% homology with the amino acid sequence shown in SEQ ID No.1 and has the same function protein.

A transgenic cell line of a coding gene of rice chloroplast protein OsFBN 7.

A recombinant strain of a coding gene of rice chloroplast protein OsFBN 7.

A recombinant vector of a coding gene of rice chloroplast protein OsFBN 7.

An application of rice chloroplast protein OsFBN7 or a coding gene thereof in improving the fatty acid content of plant chloroplasts.

An application of rice chloroplast protein OsFBN7 or a coding gene thereof in cultivating high-fat plant varieties.

A method for improving the oil content of target plants is characterized in that the coding gene of rice chloroplast protein OsFBN7 is transferred into the target plants for overexpression, and transgenic plants with the oil content higher than that of the target plants are obtained.

An application of rice chloroplast protein OsFBN7 or a coding gene thereof in regulating and controlling a rice chloroplast fatty acid de novo synthesis way.

Compared with the prior art, the invention has the advantages that:

the invention discovers that the OsFBN7 gene has the function of promoting the synthesis of lipid in chloroplasts. Experiments prove that the OsFBN7 gene is overexpressed in rice, and the obtained transgenic plant seeds show a remarkable high-fat phenotype compared with wild type. The OsFBN7 protein and the coding gene thereof can be used for breeding and identifying high-fat plant varieties in practice, and provide a new gene resource and a new method for regulating and controlling de novo synthesis of fatty acid in plant chloroplasts from sources, improving the fatty acid in the plant chloroplasts and breeding new varieties of high-fat plants.

Drawings

FIG. 1 shows PCR amplification products of OsFBN7 gene provided by an embodiment of the present invention; lane 1 shows the product band (1023 bp).

FIG. 2 is a double-restriction enzyme identification map of the OsFBN7-pMD18-T vector provided by the embodiment of the invention; the lanes are Marker bands, 1 and 2 are double-digested vector and target fragments (1023bp) of 2 positive clone plasmids.

FIG. 3 is a PCR identification chart of OsFBN7-pTCK303 colony provided in the examples of the present invention; lanes 1-5 are PCR-detected clones of the target fragment from 5 positive single colonies, which all contain 1023bp of the target fragment.

FIG. 4 is a restriction enzyme cleavage identification map of OsFBN7-pTCK303 vector provided by the embodiment of the present invention; lane M is Marker; lane 1 shows a digested band containing the target fragment (1023 bp).

FIG. 5 is a diagram showing the results of detection of OsFBN7 gene expression in overexpression strain OsFBN7 according to the present invention; wherein, WT is wild type plant of middle flower 11, OE-OsFBN7 is overexpression transgenic lines (#1, #12 and #17) of OsFBN7 gene.

FIG. 6 shows the results of lipid content detection in chloroplasts of OsFBN7 overexpression transgenic lines (OE-OsFBN 7, #17 in the figure) and wild type plants (WT-ZH 11 in the figure); lipid substances include PC, PE, PG, PI, PS, PA, MGDG, DGDG, DAG and TAG.

FIG. 7 shows the results of determination of Triglyceride (TAG) fatty acid content in mature seeds of OsFBN7 overexpression transgenic line (OE-OsFBN 7, #17) and wild type plant (WT-ZH 11); fatty acid chain lengths of triglycerides include C50:0, C50:2, C52:3, C52:4, C54:3, C54:4, and C54: 5.

FIG. 8 shows the results of testing the free fatty acid content in mature seeds of OsFBN7 overexpression transgenic lines (OE-OsFBN 7, #17) and wild type plants (WT-ZH 11); free fatty acids include C16:0, C16:1, C16:2, C18:0, C18:1, C18:2, C18:3, C20:0, C22:0, and C24: 0.

FIG. 9 is a graph showing the results of yeast two-hybrid (Y2H) identification of OsFBN7 interacting with KAS Ib, a key enzyme in the de novo fatty acid synthesis pathway in chloroplasts, provided by the example of the present invention.

FIG. 10 is a diagram showing ELISA detection results of KAS I enzyme activity in leaves of OsFBN7 overexpression transgenic lines (OE-OsFBN 7, #1, #12, #17 in the figure) and wild type plants (WT-ZH 11 in the figure) in flowering period.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The pTCK303 plasmid is commercially available in the following examples; pMD18-T is a commonly used cloning vector, commercially available; the rice variety is medium flower 11 (wild type); agrobacterium strain EHA105 genetically transformed in rice.

The main reagents in the following examples are as follows: various restriction enzymes, Taq enzyme, T4 ligase, KOD and the like are available from Bio-Inc. such as TAKARA (Dalian Blume), NEB, Toyobo and the like; dNTPs were purchased from Genestar; the plasmid miniextraction kit and the agarose gel recovery kit are purchased from Shanghai Czeri bioengineering company; MS culture medium, agar powder, agarose, antibiotics such as ampicillin (Amp), kanamycin (Kan), rifampicin (Rif) and the like, LB medium and the like are all imported or domestic analytical pure reagents.

The primers used in the following examples were synthesized by Tianyihui and subjected to correlation sequencing.

Construction and screening of rice gene OsFBN7 transgenic line

The specific method for constructing and screening the OsFBN7 gene overexpression strain is as follows:

1. the gene OsFBN7 for coding plant chloroplast protein is cloned from a rice middle flower 11 variety and is connected to an over-expression vector pTCK303 with a Ubi promoter.

Based on the coding region sequence analysis (the sequence of the OsFBN7 gene is shown as SEQ ID No. 2), a primer is designed to amplify the coding region of the OsFBN7 gene. Extracting total RNA of rice, obtaining cDNA through reverse transcription, and amplifying an OsFBN7 gene by using an upstream primer F1 and a downstream primer R1 by taking the rice cDNA as a template, wherein the amplification primers of the OsFBN7 gene are as follows:

F1：5’-ggatccgcaactcatctcggcacca-3' (as shown in SEQ ID No.3, the BamH I cleavage site is underlined);

R1：5’-ggtaccaagcgtgaaaagtggctgaac-3' (shown in SEQ ID No.4, underlined is a Kpn I cleavage site).

The OsFBN7 gene (total length of PCR product is 1023bp, as shown in FIG. 1) obtained by amplification is connected to the vector pTCK303 with pUbi promoter by the following specific method:

(1) the PCR product was ligated with pMD18-T vector using T4 ligase, the ligation product was named OsFBN7-pMD18-T (as shown in FIG. 2);

(2) the OsFBN7-pMD18-T vector is digested by BamH I and Kpn I to obtain OsFBN7, which is connected with pTCK303 vector, and the ligation product is named as OsFBN7-pTCK 303.

(3) The OsFBN7-pTCK303 plasmid obtained in the step (2) is cut by BamH I and Kpn I, and is subjected to electrophoresis at 120V and 50mA by using 1% agarose gel and scanned and imaged by an ultraviolet gel analysis system. After enzyme digestion identification is successful, sequencing the plasmid, and carrying out the next test after the sequence to be tested is correct.

Any recombinant vector, transgenic cell line and recombinant bacterium containing the gene OsFBN7 encoding the protein related to enhancing the chloroplast lipid content belong to the protection scope of the invention.

The recombinant expression vector containing the OsFBN7 gene can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pTCK303, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UBIN or other derivative plant expression vectors.

When the OsFBN7 gene is used for constructing a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added in front of the transcription initiation nucleotide, such as a cauliflower mosaic virus (CaMV)35S promoter, a Ubiquitin (Ubiquitin) gene promoter (pUbi) and the like, and the promoters can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

2. In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes expressing an enzyme or a luminescent compound which produces a color change in plants (GUS gene, GFP gene, luciferase gene, etc.), antibiotic markers having resistance (hygromycin, gentamicin, kanamycin, etc.), or chemical-resistant marker genes (e.g., herbicide-resistant gene), etc. From the safety of transgenic plants, the transformed plants can be screened directly in stress without adding any selective marker gene.

The plant expression vector carrying the plant chloroplast development related protein coding gene OsFBN7 can be transformed into target plant cells or tissues by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation and the like. The gene OsFBN7 is introduced into the target plant by the recombinant expression vector.

The OsFBN7-pTCK303 vector correctly constructed in the above example is transformed into Agrobacterium EHA105 strain by electric shock transformation method, and positive monoclonal bacteria (as shown in FIG. 3 and FIG. 4) containing OsFBN7-pTCK303 vector are obtained by identification, and then the positive monoclonal strain containing the above vector is transformed into wild rice plant to obtain rice transgenic seedling, the specific method is as follows:

(1) agrobacterium containing OsFBN7-pTCK303 vector was inoculated into 100mL YEP (added with Rif + Kan) liquid medium, and shake-cultured at 28 ℃ and 200rpm until OD of the bacterial liquid ₆₀₀ When the concentration is 0.6-1.0, centrifuging at 5000rpm for 5min, and collecting thalli; suspending the cells in AAM liquid medium containing 10mg/L AS (acetosyringone), and adjusting OD ₆₀₀ To 0.1 to 0.3;

(2) will OD ₆₀₀ Adding 0.1-0.3 AAM agrobacterium tumefaciens suspension into the dry callus with bright yellow color, soaking for 3-4 min, pouring out redundant bacteria liquid, and drying the callus; placing the callus in an N6-AS co-culture medium, and culturing for 2-3 d under the dark condition at the temperature of 22 ℃;

(3) transferring the co-cultured callus into a 50mL triangular flask, washing with sterile water, and sucking residual water with sterile absorbent paper; inoculating the callus on a selection medium containing 250mg/L carbenicillin and 50mg/L hygromycin in a mode of piling several callus together, and culturing in the dark at 28 ℃ for 2-3 weeks until new resistant callus grows out;

(4) after two times of screening, transferring the grown resistant callus to a culture medium containing regeneration MS-NK, and culturing at a normal photoperiod of 14h/10h (light/dark) and 28 ℃ until seedlings are differentiated.

The screened resistance gene carried by the pTCK303 vector is hygromycin, and the hygromycin resistance is used for screening the rice transgenic seedlings to obtain homozygous transgenic plants. The specific method comprises the following steps: subjecting the thus obtained T ₁ Carrying out single plant harvest on positive seedlings with hygromycin resistance to obtain T ₂ Generation; for T ₂ The seedlings were tested for hygromycin resistance and lines were selected 3/4 resistant and 1/4 not resistant, indicating that the overexpression vector containing the gene of interest was inserted in single copy in this line; removing the plant with hygromycin resistance from the strain, and harvesting the individual plant to obtain T ₃ Generation; for T ₃ The seedlings of the generations are screened for hygromycin resistance and homozygous transgenic lines are selected without segregation. The homozygous strain can be used for seed reproduction, chloroplast lipid content detection, enzyme activity determination, and seed lipid and fatty acid detectionContent measurement and the like.

3. And detecting the OsFBN7 gene expression condition in the obtained wild type and OsFBN7 overexpression transgenic strain OE-OsFBN7 by adopting a qRT-PCR method.

The specific method comprises the following steps: extracting total RNA of Wild Type (WT) and OsFBN7 overexpression transgenic homozygous plants, and carrying out reverse transcription to obtain cDNA; qRT-PCR detection was performed using the following primers:

f2: 5'-agcccgtcttctgtttgaga-3' (shown in SEQ ID No. 5);

r2: 5'-gaccctccacagacaactga-3' (shown in SEQ ID No. 6);

since Actin1 was detected as an internal reference gene, the primers were as follows,

f3: 5'-tgctatgtacgtcgccatccag-3' (shown in SEQ ID No. 7);

r3: 5'-aatgagtaaccacgctccgtca-3' (shown in SEQ ID No. 8);

the qRT-PCR detection result is shown in figure 5, and the result shows that the OsFBN7 gene expression quantity in the over-expression strain is obviously improved compared with the wild type. Methods for overexpressing the OsFBN7 gene in other plants can be performed with reference to this example.

Detection of lipid content in chloroplast of overexpression OsFBN7 transgenic rice line

The specific method for detecting the chloroplast lipid content in the OsFBN7 overexpression transgenic rice homozygous line obtained in the above embodiment is as follows:

5-10 g of a well-grown OsFBN7 overexpression transgenic homozygous line and 5-10 g of wild type (ZH11) rice leaves are selected, and complete chloroplasts are extracted by a Percoll gradient preparation method.

Taking 0.5-1 mg chloroplast in a 50mL centrifuge tube, adding 10mL methanol, 10mL chloroform and 5mL ddH ₂ The solution was vortexed to make it cloudy, centrifuged at 1500g for 5min, the lower chloroform phase (lipid) was removed, the solvent was evaporated on a low pressure rotary evaporator, 250. mu.L of chloroform was dissolved, and lipid content measurement (GC-MS) was performed.

The results of the lipid content measurement are shown in FIG. 6. The results show that the contents of Diglyceride (DAG) and glycolipid (MGDG and DGDGDG) in chloroplasts of the OsFBN7 gene overexpression transgenic homozygous lines are obviously increased compared with the wild type.

Detection of lipid and fatty acid content in overexpression OsFBN7 transgenic rice seeds

The method for extracting and measuring the grease in the mature seeds comprises the following steps:

after the ripe rice seeds are ground in a mill, the rice powder (100g) is mixed with 300ml chloroform/methanol (2:1, v/v) evenly, the mixture is extracted for 2 hours by gentle shaking, and the extraction is repeated for three times. Then, 20ml of KCl (0.75%) was added to the extract, and the chloroform layer was extracted and concentrated in vacuo in a rotary evaporator. The lipids were then transferred to a 25ml brown glass bottle and chloroform/methanol (2:1, v/v) was added. After blowing-dry with nitrogen, the cells were stored at-25 ℃ for subsequent analytical determination. The extracted lipids are subjected to methyl esterification reaction to prepare free fatty acid methyl ester components, and the ESI-MS/MS is utilized to determine the content of TAG (triglyceride) and fatty acid in mature rice seeds.

The measurement results of the TAG content and the free fatty acid content in the mature rice seeds are shown in FIGS. 7 and 8.

The results in FIG. 7 show that the content of TAG in mature seeds of OsFBN7 gene overexpression transgenic homozygous lines is obviously increased compared with that of wild type. The average content of TAG in the OsFBN7 overexpression transgenic line is 17.5 mu mol ^-1 DW (dry weight seed), 11 flowers (14.1. mu. mol. mg) in comparison with wild type ^-1 DW) was increased by 24.1%. This indicates that OsFBN7 promotes the synthesis and accumulation of oil bodies (TAGs) in mature seeds. Wherein, the overexpression of OsFBN7 obviously improves the composition and content of C52:2, C52:3, C52:4 and C54:3 oil body fatty acid.

The results of FIG. 8 show that the content of free fatty acids C16:0, C18:0, C18:1 and C18:2 in the mature rice seeds is significantly higher than that of flower 11 in the wild type. Furthermore, the fatty acid species and free fatty acid composition in TAG are identical. Therefore, OsFBN7 promotes the synthesis and accumulation of free fatty acids in mature rice seeds.

OsFBN7 protein and KAS IbIdentification of interaction

The identification method of the point-to-point yeast two-hybrid (Y2H) of the interaction between OsFBN7 and OsKAS Ib is as follows:

there are 2 KAS I (β -ketoacyl-ACP synthase I) genes in rice: OsKAS Ia and OsKAS Ib, and the homology of the two genes coding proteins is as high as 84.5%, and the two genes possibly have similar functions. Therefore, on the basis of the screening result of the yeast two-hybrid library, in order to verify the interaction between OsFBN7 and OsKAS I b protein, the invention constructs an OsKAS Ib-pGADT7 plasmid and an OsFBN7-pGBKT7 plasmid, and carries out point-to-point yeast two-hybrid verification.

1. The yeast plasmid OsKAS Ib-pGADT7 of the candidate gene was transformed into Escherichia coli JM109, and the Escherichia coli plasmid containing the candidate gene was extracted.

2. The recombinant plasmid of the escherichia coli plasmid OsKAS Ib-pGADT7 and OsFBN7-pGBKT7 containing the candidate gene is transformed into the yeast AH109 by a cotransformation method.

3. The recombinant plasmid OsKAS Ib-pGADT7 or OsFBN7-pGBKT7 containing the target gene is transformed into yeast AH109 separately.

4. Transformed yeast AH109 was plated on auxotrophic selection medium containing two deletions (SD-Trp-His), three deletions (SD-Trp-Ade-His) and four deletions (SD-Trp-Leu-His-Ade + α -X-gal) at a concentration of 20mg/L α -X-gal, respectively. If the co-transformed yeast grows normally on medium lacking four (SD-Trp-Leu-His-Ade + alpha-X-gal) and the colonies turn blue, then the yeast two-hybrid validated candidate interacting protein gene can be considered. The results in FIG. 9 show that there is indeed an interaction between OsKAS Ib and OsFBN7 proteins in rice chloroplasts.

Determination of KAS I enzyme activity in OsFBN7 overexpression strain leaves

On the basis of identifying the interaction between OsFBN7 and OsKAS Ib, the invention determines the KAS I enzyme activity in the leaves of OsFBN7 overexpression transgenic homozygous lines, and the specific method is as follows:

and selecting a well-grown OsFBN7 overexpression transgenic homozygous strain and 1g of wild type ZH11 rice flowering stage flag leaves, and extracting with enzyme solution.

The enzyme activity of KAS I is detected by utilizing an enzyme-linked immunoassay kit of beta-ketoacyl-ACP synthase I of Wuhan dynasty Bory company. 4 biological replicates per sample were performed.

And drawing a standard curve by using Excel and calculating a regression equation of the standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate. Substituting the OD value of the sample into an equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.

The detection result of the KAS I enzyme activity in the rice leaves is shown in figure 10, and the result shows that the overexpression of OsFBN7 obviously improves the KAS I enzyme activity in the leaves. Thus, this experiment fully demonstrates the interaction between OsFBN7 and OsKAS Ib. Furthermore, the enzymatic activity of OsKAS I was significantly increased in OsFBN7 overexpression transgenic homozygous lines compared to wild type. OsFBN7 was shown to enhance the de novo fatty acid biosynthesis pathway in rice chloroplasts by promoting the enzymatic activity of OsKAS I. Finally, the content of fatty acid in the mature rice seeds is improved.

In summary, the de novo fatty acid synthesis pathway in plant chloroplasts is the major fatty acid synthesis pathway in plants. The initial step of the de novo fatty acid synthesis pathway is catalyzed by acetyl-coa carboxylase (ACCase). Then, condensation by three types of β -ketoacyl-ACP synthases (KAS III, I, II) results in staged elongation of the fatty acid carbon chain. The role of these KAS proteins in the condensation and extension of the fatty acid carbon chain from C2 to C18 in plant chloroplasts is crucial. Currently, there are 5 KAS proteins in rice chloroplasts: including OsKAS Ia/Ib, OsKAS IIa/IIb and OsKAS III. OsKASIII is mainly responsible for the extension of carbon chains C2-C4, OsKAS I is mainly responsible for the extension of C4-C16 of fatty acids, and OsKAS II is mainly responsible for the extension of C16-C18 of fatty acids. Therefore, it plays a very critical role in the de novo biosynthesis of fatty acids. The invention proves that the OsFBN7 protein can promote the enzyme activity of OsKAS I by interacting with OsKAS Ib protein, thereby improving the content of lipid in plant chloroplast. Finally, the contents of grease and fatty acid in the mature rice seeds are obviously improved, and the results provide a new idea and a new way for cultivating the high-grease plant seeds. Therefore, in production, the gene OsFBN7 for regulating the de novo biosynthesis of fatty acid in rice is utilized to provide a characteristic and application potential gene resource for the cultivation of plant varieties with high oil content. The gene plays an important role in the research of improving the content of vegetable oil and fat and regulating the synthesis way of fatty acid by using a genetic engineering means, and has important practical value and economic benefit.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> rice chloroplast protein OsFBN7 and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 301

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Ala Ala Ala Ala Ala Ala Ala Leu Ile Thr Ala Ala Ser Thr Pro

1 5 10 15

Phe Pro Leu Val Ser Phe Arg Ser Arg Arg Asp Gly His Leu Ser Leu

20 25 30

Ser Pro Pro Arg Arg Pro Gly Ala Gly Arg Cys Arg Ala Ser Ala Pro

35 40 45

Thr Phe Gln Gly Gly Pro Ala Ala Ser Tyr Ala Arg Glu Met Glu Arg

50 55 60

Leu Ser Ala Lys Glu Ser Leu Leu Leu Ala Phe Arg Asp Ala Gly Gly

65 70 75 80

Phe Glu Ser Leu Val Ser Gly Lys Thr Thr Gly Met Gln Lys Ile Asp

85 90 95

Val Asn Glu Arg Ile Val Gly Leu Glu Arg Leu Asn Pro Thr Pro Arg

100 105 110

Pro Thr Thr Ser Pro Phe Leu Glu Gly Arg Trp Asn Phe Glu Trp Phe

115 120 125

Gly Asp Ser Ser Pro Gly Ala Leu Ala Ala Arg Leu Leu Phe Glu Arg

130 135 140

Ser Pro Thr Thr Val Ala His Phe Thr Gly Leu Asp Val Leu Ile Lys

145 150 155 160

Asp Gly Tyr Ser Lys Ile Ser Ser Asn Val Lys Phe Leu Asn Thr Val

165 170 175

Gln Ser Lys Phe Leu Leu Thr Thr Gln Leu Ser Val Glu Gly Pro Ile

180 185 190

Arg Met Lys Glu Glu Tyr Val Glu Gly Leu Ile Glu Ile Pro Arg Ile

195 200 205

Arg Glu Glu Thr Leu Pro Asp Gln Leu Lys Gly Phe Phe Gly Gln Thr

210 215 220

Ala Gly Ala Leu Gln Gln Leu Pro Ala Pro Ile Arg Asp Ala Val Ser

225 230 235 240

Glu Gly Ile Lys Leu Pro Leu Asn Gly Met Phe Gln Arg Leu Phe Met

245 250 255

Ile Ser Tyr Leu Asp Glu Glu Ile Leu Ile Ile Arg Asp Ala Ser Gly

260 265 270

Ala Pro Asp Val Leu Thr Arg Leu Glu Gly Pro Gln Pro Asn Ser Ile

275 280 285

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

290 295 300

<210> 2

<211> 906

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggccgccg ccgccgccgc ggcgctcatc accgccgcct ccactccttt cccgctggtc 60

tccttccgca gccggcggga tggccacctg agcctttcgc ctcctcgccg ccccggcgcc 120

gggcgctgca gggcctcggc gccgacgttc caggggggac ccgccgccag ctacgcccgc 180

gagatggagc gcctctccgc caaggagtcc ctcctcctcg ccttcagaga tgctggaggg 240

tttgaatcct tggttagtgg aaagactaca gggatgcaga agattgatgt caatgagcgg 300

attgttgggc tcgagcgtct aaaccccact ccacggccca caacatctcc ctttctggaa 360

ggtcgatgga actttgaatg gtttggtgac agcagtcctg gagcacttgc agcccgtctt 420

ctgtttgaga ggtcacctac aactgttgca cactttacag gacttgatgt cctgatcaag 480

gatggatact ccaaaatttc ttccaacgtg aagttcttaa atacggtgca aagcaagttc 540

ctgctgacta ctcagttgtc tgtggagggt cctattagga tgaaagagga atatgtcgag 600

ggcctaatag agatccctag gatcagagaa gaaacactgc ctgatcaact aaagggtttc 660

tttggacaaa ctgcaggtgc tctgcaacaa ctccctgccc ccataaggga cgctgtttca 720

gaggggatta aattgccact taatgggatg ttccagcgcc tattcatgat ttcttacttg 780

gatgaggaaa tactgattat cagagatgca tctggagcac ctgatgtcct aacaagattg 840

gaagggccac agccaaattc aattgatggc acatcagacg cagtgctgtc agaatatgaa 900

agctag 906

<210> 3

<211> 25

<212> DNA

<213> F1(Artificial Sequence)

<400> 3

ggatccgcaa ctcatctcgg cacca 25

<210> 4

<211> 27

<212> DNA

<213> R1(Artificial Sequence)

<400> 4

ggtaccaagc gtgaaaagtg gctgaac 27

<210> 5

<211> 20

<212> DNA

<213> F2(Artificial Sequence)

<400> 5

agcccgtctt ctgtttgaga 20

<210> 6

<211> 20

<212> DNA

<213> R2(Artificial Sequence)

<400> 6

gaccctccac agacaactga 20

<210> 7

<211> 21

<212> DNA

<213> F3(Artificial Sequence)

<400> 7

gctatgtacg tcgccatcca g 21

<210> 8

<211> 22

<212> DNA

<213> R3(Artificial Sequence)

<400> 8

aatgagtaac cacgctccgt ca 22

Claims (5)

1. The application of the rice chloroplast protein OsFBN7 or the coding gene thereof in regulating and controlling a rice chloroplast fatty acid de novo synthesis way is characterized in that the amino acid sequence of the rice chloroplast protein OsFBN7 is shown as SEQ ID No. 1.

2. The application of the rice chloroplast protein OsFBN7 or the coding gene thereof in regulating and controlling a rice chloroplast fatty acid de novo synthesis way is characterized in that the nucleotide sequence of the rice chloroplast protein OsFBN7 coding gene is shown as SEQ ID No. 2.

3. An application of a rice chloroplast protein OsFBN7 or a coding gene thereof in improving the fatty acid content of rice chloroplasts in an overexpression mode, wherein the amino acid sequence of the rice chloroplast protein OsFBN7 is shown as SEQ ID No. 1.

4. An application of a rice chloroplast protein OsFBN7 or a coding gene thereof in breeding high-fat rice varieties in an overexpression mode, wherein an amino acid sequence of the rice chloroplast protein OsFBN7 is shown as SEQ ID No. 1.

5. A method for improving the oil content of target rice is characterized in that a coding gene of rice chloroplast protein OsFBN7 is transferred into the target rice for overexpression to obtain a transgenic plant with the lipid content higher than that of the target rice, wherein the amino acid sequence of the rice chloroplast protein OsFBN7 is shown as SEQ ID No. 1.

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