A transcription factor NnMYB33 gene related to starch synthesis in lotus rhizomes and its application

Abstract

The present invention discloses a transcription factor NnMYB33 gene related to starch synthesis in lotus rhizomes and its application. Starch is the main storage material of lotus rhizomes, accounting for more than 70% of the dry matter weight of rhizomes; the content and components of starch directly determine the yield and edible quality of lotus rhizomes, and are indicators reflecting the quality of lotus processed products. The present invention clones a new NnMYB33 gene from lotus roots. The present invention uses genetic engineering technology to overexpress the NnMYB33 transcription factor in potatoes, which can significantly increase the starch content of transgenic plants. The present invention uses RT-PCR technology to clone a NnMYB33 gene from lotus roots, which can increase the starch content of plants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a transcription factor NnMYB33 gene related to lotus rhizome starch synthesis and an application thereof. Background Art

[0002] Lotus root (Nelumbo nucifera Gaertn.) is an aquatic herbaceous plant belonging to the genus Nelumbo of the family Nelumbo. It is the largest aquatic vegetable cultivated in China. The lotus root rhizome is rich in starch, flavonoids, alkaloids and multiple vitamins, and has important medicinal values ​​such as warming and nourishing, clearing the lungs, etc. It is an important "medicine and food" type vegetable.

[0003] Starch is the main storage material of lotus rhizomes, accounting for more than 70% of the dry matter weight of rhizomes; the content and composition of starch directly determine the yield and edible quality of lotus rhizomes, and are indicators of the quality of lotus processed products. Starch is a natural polymer linked by glucose. According to the differences in polymer chain length and branching, starch is divided into amylopectin and straight-chain starch. According to the starch content and composition of lotus root products, lotus roots are divided into two categories: crisp and powdery. Among them, the starch content of crisp lotus roots generally accounts for less than 15% of the fresh weight; the starch content of powdery lotus roots generally accounts for more than 17% of the fresh weight. The biosynthesis of starch is a complex metabolic process that requires the participation of multiple enzymes, which mainly include 5 enzymes, namely 1,6-adenosine diphosphate glucose pyrophosphorylase, granule-bound starch synthase, starch synthase, starch branching enzyme and starch debranching enzyme. In addition, plant hormones, protein kinases and transcription factors play an important regulatory role in starch biosynthesis. Since lotus roots have been propagated asexually for a long time, the genetic traits of starch and other plants are narrow, making it difficult to breed high-quality new lotus root varieties through traditional hybridization methods. Clarifying the regulatory mechanism in starch biosynthesis will provide a scientific basis for the research and application of lotus root quality genetic engineering, and provide theoretical guidance for the use of molecular breeding methods to breed high-quality new lotus root varieties.

[0004] Transcription factors are important regulatory elements in the process of plant growth and development. Among them, MYB transcription factors, as the largest class of transcription factors in plants, play a key role in quality regulation and response to biotic and abiotic stresses. MYB transcription factors are involved in the regulation of plant phenylpropanoid secondary metabolic pathways and are closely related to the regulation of starch synthesis. For example, wheat transcription factor TaMYB44 can regulate starch biosynthesis by activating the expression of starch synthesis-related genes. In addition, MaMYB16L and MaMYB33 in bananas negatively regulate the expression of starch degradation-related genes, thereby affecting starch degradation in bananas. The identification of excellent quality regulatory genes of lotus root can provide an important scientific basis for the cultivation of high-quality lotus root varieties through modern molecular breeding. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art. The present invention provides a transcription factor NnMYB33 gene related to starch synthesis in lotus rhizomes and its application.

[0008] The gene encodes the lotus root MYB transcription factor NnMYB33, which can promote the activity of the promoter of the key gene NnSS1 in the starch biosynthesis pathway, positively regulate the expression of NnSS1, and thus promote the biosynthesis of starch; using genetic engineering technology to overexpress the NnMYB33 transcription factor in potatoes can significantly increase the starch content of transgenic plants, which is of great significance for the germplasm innovation of lotus roots and the breeding of high-content starch varieties. Therefore, the purpose of the present invention is to provide an application of a protein NnMYB33 derived from lotus roots in regulating the quality of plant starch.

[0009] In the first aspect, the present invention claims the use of NnMYB33 protein or its related biological materials in regulating plant starch biosynthesis; the related biological materials are nucleic acid molecules capable of expressing the NnMYB33 protein or expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules;

[0010] The NnMYB33 protein is any of the following proteins:

[0011] (A1) a protein consisting of the amino acid sequence shown in SEQ ID No. 1;

[0012] (A2) a protein derived from SEQ ID No. 1, wherein one or more amino acid residues are substituted and / or deleted and / or added and which has the same function;

[0013] (A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

[0014] (A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any one of (A1) to (A3).

[0015] (A5) The protein shown in SEQ ID NO.1 is encoded by the gene shown in SEQ ID NO.2.

[0016] The NnMYB33 protein is derived from lotus root.

[0017] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0018] In the above proteins, the tag may be a Flag tag, a His tag, a MBP tag, a HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0019] The application is specifically embodied in that the activity and / or expression level of the NnMYB33 protein or the nucleic acid molecule capable of expressing the NnMYB33 protein in the plant is increased, and the starch content of the plant is increased.

[0020] In a second aspect, the present invention provides a method for protecting and cultivating plant varieties with increased starch content, comprising the step of increasing the expression level and / or activity of NnMYB33 protein in a recipient plant; the NnMYB33 protein is any one of the proteins shown in (A1)-(A4) above.

[0021] Furthermore, the present invention claims a method for cultivating transgenic plants, comprising the following steps:

[0022] A nucleic acid molecule capable of expressing NnMYB33 protein is introduced into a recipient plant to obtain a transgenic plant; the starch content of the transgenic plant is higher than that of the recipient plant, and the accumulation of starch leads to the swelling of the edible part of the transgenic plant, etc. The "introducing a nucleic acid molecule capable of expressing NnMYB33 protein into the recipient plant" is achieved by introducing a recombinant expression vector containing a nucleic acid molecule capable of expressing NnMYB33 protein into the recipient plant.

[0023] Existing expression vectors can be used to construct a recombinant expression vector containing the coding gene of the NnMYB33 protein. The plant expression vector includes a binary Agrobacterium vector and the like.

[0024] In order to facilitate the identification and screening of transgenic plant cells or plants, reporter gene tags can be added to the vector used, such as the green fluorescent protein tag gene (GFP gene), kanamycin resistance protein tag gene, hygromycin resistance protein tag gene, etc.

[0025] In the present invention, the recombinant expression vector may specifically be a recombinant plasmid obtained by inserting a DNA fragment represented by nucleotides 1 to 1674 of the NnMYB33 nucleic acid molecule sequence from the 5' end into the pCAMBIA1301 vector.

[0026] In a third aspect, the present invention claims protection for the above-mentioned NnMYB33 protein or its related biological materials, or the use of any of the above-mentioned methods in plant breeding.

[0027] The aim of such breeding is to select plants with an increased starch content.

[0028] Experiments have shown that the NnMYB33 of the present invention can increase the starch content in plants, can be used to prepare products for increasing the starch content of plants, and can be directly used to improve the starch quality of plants, providing an important scientific basis for cultivating high-quality and high-yield new varieties using modern molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0030] Figure 1 It is a sequence comparison analysis of the lotus root NnMYB33 protein in the present invention and the MYB transcription factors in other species;

[0031] Figure 2 This is a schematic diagram of the expression detection and analysis of the lotus root NnMYB33 gene in different tissues of lotus root;

[0032] Figure 3 This is a schematic diagram of the relative expression amount obtained by converting the fluorescence intensity in Example 3;

[0033] Figure 4 Schematic diagram of agarose gel electrophoresis of NnMYB33 gene in Example 4;

[0034] In the figure, the fragment size of the band concentration is 1677 bp;

[0035] Figure 5 Determination of starch content in NnMYB33 overexpression and control groups;

[0036] Figure 6 Electron microscopic examination of starch granules in NnMYB33 overexpression and control groups;

[0037] Figure 7 This is a schematic diagram of the relative expression level obtained by converting the fluorescence intensity in Example 3. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0039] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0040] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0041] The 'MRH' used in the embodiments of the present invention is a variety of Meirenhong that is widely planted in Jiangsu Province.

[0042] Example 1

[0043] This example is used to illustrate how to prepare a template for detecting gene expression:

[0044] Extraction of total RNA from lotus root and synthesis of cDNA: RNA from mature rhizomes of 'MRH' lotus root was extracted using the Plant Total RNA Rapid Extraction Kit (ZP405) from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd. TM The cDNA was synthesized using the 1st Strand cDNA Synthesis Kit (6110A) and used as a template for detecting the expression of genes related to starch synthesis.

[0045] Example 2

[0046] This example is used to construct a fluorescent quantitative PCR system:

[0047] The NnMYB33 gene-specific primer pair (NnMYB33DLF and NnMYB33DLR) was designed using the online tool in the NCBI website. The cDNA in Example 2 was used as a template and the fluorescence quantitative kit ChamQ UniversalSYBR qPCR Master Mix of Novozymes was used for detection. The detection system is shown in Table 1:

[0048] Table 1 Fluorescence quantitative PCR reaction system

[0049]

[0050] The PCR program was set as follows:

[0051]

[0052] The fluorescence signal intensity of MYB33 in different tissues of lotus root was detected 80 days after lotus root planting. －△△ C T The relative expression level of MYB33 was obtained by the method. Figure 2 shown.

[0053] Depend on Figure 2 It can be seen that MYB33 has the best amplification effect in the early stage of lotus root rhizome expansion, indicating that MYB33 plays an important role from the beginning.

[0054] In addition, through the analysis of the transcriptome of different lotus root tissues, it was found that MYB33 was highly expressed in the lotus root rhizome, indicating that MYB33 mainly plays a role in the lotus root rhizome, such as Figure 3 shown.

[0055] Example 3

[0056] This example is used to clone, verify and recover the target gene:

[0057] Cloning of NnMYB33 gene: A primer pair (NnMYB33F and NnMYB33R) was designed based on the sequence of NnMYB33 gene, NnMYB33F and NnMYB33R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. The NnMYB33 gene was amplified by PCR using the lotus root cDNA prepared in Example 1 as a template.

[0058] NnMYB33F: 5'-ATGGGTCGTTCAACAAATGA-3';

[0059] NnMYB33R: 5'-TCAAGGGAGTTCAGACATATGACAAACC-3'.

[0060] The PCR amplification product was subjected to 1.2% agarose gel electrophoresis, and a band of about 1677 bp was recovered and purified using a common agarose gel DNA recovery kit (DP209 from Tiangen Biochemical Technology Co., Ltd.).

[0061] The obtained bands are Figure 4 shown.

[0062] Depend on Figure 4 It can be seen that NnMYB33F: and NnMYB33R can play an obvious effect on the cloning of the target gene, and the bands are concentrated in the same position and have a high brightness, that is, the purity of the target gene in our invention is high and the amount of the target gene obtained is large.

[0063] Example 4

[0064] This example is used to transfer the target gene to produce transgenic potato plants:

[0065] Obtaining transgenic potato plants: The full-length sequence of NnMYB33 gene was cloned, connected to the pCAMBIA1301 overexpression vector (the vector carries the 35S CaMV promoter sequence and the kanamycin selection marker gene KanR) and transformed into Agrobacterium strain GV3101. Potato chips of 'Eshu No. 3' pre-cultured in callus induction medium for about 15 days were selected and placed in a conical flask containing Agrobacterium bacterial solution for shaking and co-culture for about 10 minutes; the bacterial solution was dried with sterile filter paper, and the callus was placed in a callus induction medium for co-culture in the dark (28°C) for about 3 days, washed with sterile water 3 times, and then transferred to a callus induction medium containing 600 mg / L cephalosporin (Cef) and 50 mg / L kanamycin (Kan) for continued cultivation; the green callus with larger induced growth was transferred to a callus differentiation medium containing 600 mg / L Cef and 50 mg / L Kan for cultivation; after a few days, the adventitious buds obtained were transferred to MS medium containing 600 mg / L Cef and 50 mg / L Kan for rooting; when potato seedlings grew out, they were divided into NnMYB33 according to the strains. OE-1 to 10, DNA and RNA were extracted for transgenic identification, and positive seedlings were obtained. The obtained positive seedlings were divided into the following according to different strains: NnMYB33 OE-3, NnMYB33 OE-7, NnMYB33 OE-8, such as Figure 5 shown.

[0066] Depend on Figure 5 A potato plant containing the target gene was obtained.

[0067] Example 5

[0068] This example is used to verify the performance of transgenic plants:

[0069] Determination of starch content in transgenic plants: Take samples of transgenic plants and control plants (potato tubers) and sterilize them in an oven at 105°C for 1 hour, then dry them at a constant temperature of 60°C to constant weight, crush and dry them, and collect the powder for later use. 80% ethanol can be used to separate the soluble sugar and starch in the sample, and the starch can be further decomposed into glucose by acid hydrolysis. The glucose content is determined by the anthrone colorimetric method, and the total starch content of lotus root can be calculated. For specific methods, please refer to the instructions for the starch content detection kit of Solebaud (BC0700). The measured data is as follows: Figure 6 As shown. Electron microscope scanning of starch granules revealed Figure 7 .

[0070] Depend on Figure 6It can be seen that the starch content of transgenic plants was significantly increased compared with the control group.

[0071] The amylopectin content is detected by using the property of amylopectin and iodine to form a red-purple complex. Ethanol is used to separate the soluble sugar and starch in the sample, and then iodine is used to react with them to obtain the amylopectin content. For specific detection operation methods, please refer to the instructions for the amylopectin content detection kit of Solebo Company (BC4270).

[0072] Table 2 Comparison of starch content between transgenic potatoes and wild-type potatoes

[0073]

[0074] "*" indicates significant correlation, "NS" indicates no correlation;

[0075] As shown in Table 2, in terms of total starch content, the transgenic potatoes have a more significant increase in content compared to the wild-type potatoes. While there is no significant difference in the content of amylose, the content of amylopectin has been significantly increased.

[0076] Example 6

[0077] This example is used to observe the starch morphology of transgenic plants:

[0078] Observation of starch granule morphology of transgenic plants: Gather transgenic and control materials with the same growth potential, collect samples after treatment and store them in a desiccator for later use. Dilute the starch sample with anhydrous ethanol to form a suspension, absorb an appropriate amount of the suspension on the sample stage, dry the sample stage, place it in a particle sputtering plating instrument for gold plating, and then observe it under an environmental scanning electron microscope (Philips XL-30). For each treated sample, 15 starch granule morphology pictures were randomly selected, and the long and short axes of the starch granules in the field of view were measured using the ruler of the electron microscope, and the data were recorded and statistically analyzed. The data obtained were recorded in Figure 7 middle.

[0079] Depend on Figure 7 It can be seen that the total number of starch granules in transgenic potatoes overexpressing NnMYB33 is significantly higher than that in wild-type potatoes, indicating that NnMYB33 can promote starch accumulation by promoting the increase in the number of starch granules in transgenic plants.

[0080] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A transcription factor overexpressed and associated with starch synthesis in lotus rhizomes NnMYB33 The application of genes is characterized by: Said NnMYB33 The gene sequence is shown in Seq ID No.

2. NnMYB33 Overexpression of the gene significantly increased the amylopectin content.

2. The use according to claim 1, characterized in that: Said NnMYB33 The gene length is 1677bp. NnMYB33 The number of amino acids encoded in the gene is 558.

3. The use according to claim 1, characterized in that: Said NnMYB33 Overexpression of the gene resulted in higher total starch and amylopectin contents than those of the wild type.

4. The use according to claim 1, characterized in that: Said NnMYB33 The overexpression vector for gene overexpression was pCAMBIA1301.

5. The use according to claim 1, characterized in that: include, Select plant material: NnMYB33 The gene source plant and the recipient plant into which it is ultimately transformed; Extraction of total RNA and synthesis of cDNA: Extract total RNA from the source plant and convert the total RNA into cDNA; NnMYB33 Gene cloning: Design cloning primers, use PCR to clone the cDNA obtained by reverse transcription as a template, and connect it to the vector; Sequence analysis: Sequencing comparison of lotus root NnMYB33 The amino acid sequence encoded by the gene; qPCR amplification: Design primers for fluorescence quantitative PCR test to detect NnMYB33 The expression level of genes at different developmental stages; Construction of vector and transformation into recipient plant cells: Design of primers to achieve homologous recombination NnMYB33 The gene is connected to a vector, and the recombinant vector is transformed into recipient plant cells; Starch expression: The transformed strains are subjected to starch expression.

6. The use according to claim 1 or 5, characterized in that: Said NnMYB33 In the gene cloning, the cloning primers were forward primer: 5'-ATGGGTCGTTCAACAAATGA-3'; reverse primer: 5'-TCAAGGGAGTTCAGACATATGACAAACC-3'.

7. The use according to claim 5, characterized in that: The construct vector is transformed into a recipient plant cell, and the vector is a pCAMBIA-1301 vector.

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