CN117210463B - miRNA related to lotus root rhizome starch synthesis and application thereof - Google Patents
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Abstract
The invention discloses miRNA related to lotus root rhizome starch synthesis and application thereof, which comprises the following steps: the gene sequence of miRNA is shown in Seq ID No. 1. The miR395b can reduce the starch content in plants, can be used for preparing products for reducing the starch content in plants and can be directly used for improving the starch quality of the plants, and an important scientific basis is provided for cultivating new varieties with high quality and high yield by utilizing modern molecular breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to miRNA (micro ribonucleic acid) related to lotus root rhizome starch synthesis and application thereof.
Background
Lotus root (Nelumbo nucifera Gaertn.) is perennial herbaceous plant of lotus genus of lotus family, is the biggest characteristic aquatic vegetable of planting area in our country, has important scientific research and economic value. The fresh lotus root and the processed products thereof have rich nutritional value and unique health care function, are rich in starch, protein, amino acid, polysaccharide, polyphenol, alkaloid and mineral elements, and are deeply favored by consumers.
Starch is a renewable and biodegradable natural polymer substance, is a polysaccharide polymerized by glucose molecules, and consists of amylose and amylopectin. The starch content is about 70% or more of the dry matter mass of lotus root, is the main storage substance of lotus root, and plays a very important role in the plant growth process. The enlarged rhizome is a main edible organ of lotus root, starch is an important component of lotus root rhizome, the synthesis of starch is regulated and controlled by various enzymes, and the physical and chemical properties of starch are determined by the total starch, the composition of amylose and amylopectin and the hierarchical fine structure formed by the starch, so that the yield and the edible quality of the lotus root are affected. Because the lotus roots are asexually propagated for a long time, the genetic characters of starch and the like are narrow, and it is difficult to cultivate high-quality new lotus root varieties by the traditional hybridization method. The regulation and control mechanism in the biosynthesis of starch is clarified, scientific basis is provided for the genetic engineering research and application of lotus root quality, and theoretical guidance is provided for cultivating a new variety of high-quality lotus root by utilizing a molecular breeding means.
miRNA (microRNA) is a small molecular RNA with the length of about 18-24nt, has high conservation in evolution, and recent researches show that various biological processes of plants are regulated and controlled by miRNA, including growth and development of plants, morphogenesis, signal transduction, response to biotic and abiotic stress and the like. Meanwhile, miRNA has close relation with starch synthesis regulation, for example, the mRNA level of transcription factors ZmMYB138 and ZmMYB115 is negatively regulated by maize miR159a so as to influence the expression of key corn endosperm starch synthesis enzyme genes ZmSBE2b, zmBt2 and ZmGSS, and finally, the biosynthesis of starch is reduced. miR169o affects starch biosynthesis in corn endosperm by negatively regulating target gene SS3a, SBE2 b. The identification of excellent quality regulation miRNA of lotus roots can provide important scientific basis for breeding high-quality lotus root varieties in modern molecular breeding.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide miRNA related to lotus root rhizome starch synthesis.
In order to solve the technical problems, the invention provides the following technical scheme: a miRNA associated with lotus root-like stem starch synthesis, comprising: the gene sequence of miRNA is shown in Seq ID No. 1.
As a preferred embodiment of the miRNA related to lotus root rhizomes starch synthesis of the present invention, the miRNA is: the miRNA has a length of 135bp.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis.
In order to solve the technical problems, the invention provides the following technical scheme: an application of miRNA related to lotus root-like stem starch synthesis, comprising: transferring miRNA into other plants for overexpression,
another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: and transferring miRNAs into other plants for overexpression.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: expression of mirnas reduced the content of amylopectin and amylose.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: the expression of miRNA decreased amylopectin content by an extent greater than amylose.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: miRNA is expressed in Arabidopsis, rice, maize, tomato, tobacco.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising the following steps:
selecting plant material: extracting RNA of lotus root tubers in the mature period, namely miRNA;
cloning, acceptance and recovery of mirnas: designing a primer pair for miRNA, amplifying, electrophoresis and recovering;
transferring target genes and preparing transgenic plants: transforming the target gene into microorganism and infecting plant, and identifying to obtain positive seedling;
expression and detection: positive seedlings were subjected to starch expression and to detection of starch expression level.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: in the cloning, acceptance and recovery of miRNA, the primer pair is as follows
miR395bF:5'-GACTCTGCAGGTCGAATCATCAGGTGTCCCCTAGA-3'；
miR395bR:5'-GATCTCTAGAGTCGAATAAAATCCAGCGGCCC-3'。
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: the target gene is transferred into the transgenic plant, and the microorganism is agrobacterium strain GV3101.
Another object of the present invention is to provide an application of miRNA related to lotus root rhizome starch synthesis, comprising: transferring target gene and preparing transgenic plant, the infected plant is tobacco callus cultured by infection callus induction
The invention has the beneficial effects that:
the miR395b can reduce the starch content in plants, can be used for preparing products for reducing the starch content in plants and can be directly used for improving the starch quality of the plants, and an important scientific basis is provided for cultivating new varieties with high quality and high yield by utilizing modern molecular breeding.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a sequence alignment analysis of lotus root miR395b in the present invention with mature sequences in other species;
FIG. 2 is a schematic diagram of expression detection analysis of a miR395b gene of lotus roots in different periods of the lotus roots;
FIG. 3 is a schematic diagram showing agarose gel electrophoresis of miR395b gene in example 4;
in the figure, the size of the band concentrated fragment is 136bp;
FIG. 4 is a phenotypic analysis of transgenic tobacco over-expressing miR395 b;
FIG. 5 is a starch assay of miR395b over-expression and a control group;
fig. 6 is a starch chain length assay of miR395b over-expression and control.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Seq ID No.1：
ATCATCAGGTGTCCCCTAGAGTTCCCTCAACCCCTTCAGTGGGACTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGAGTTCAAGCTTTCCCTCCTACTGAAGTGTTTGGGGGAACTCCGGGGGCCGCTGGATTTTAT
Seq ID No.2：GACTCTGCAGGTCGAATCATCAGGTGTCCCCTAGA；
Seq ID No.3：GATCTCTAGAGTCGAATAAAATCCAGCGGCCC。
Example 1
This example is for explaining how to prepare a template for gene expression level detection:
extracting lotus root total RNA and synthesizing cDNA: and (3) extracting RNA of the lotus root rhizome in the maturation period of 'MRH' by referring to a rapid extraction kit (ZP 405) of plant total RNA of Beijing bang nationality biological gene technology limited company, namely miRNA, wherein the nucleic acid sequence of the RNA is shown as a Seq ID No. 1. According to PrimeScript from TaKaRa TM 1st Strand cDNA Synthesis Kit (6110A) method is used for synthesizing cDNA, which is used as a template for detecting the expression level of genes related to starch synthesis.
The sequence alignment analysis of the lotus root miR395b obtained in the process and mature sequences in other species is shown in a figure 1, and other sequences in the figure are respectively obtained from Arabidopsis thaliana, rice, corn and tomatoes in sequence from top to bottom.
Example 2
The present example was used to perform fluorescent quantitative PCR system construction:
gene-specific primer design was performed on miR395b using primer design software DNAMAN, and detection was performed using the cDNA in example 1 as a template and a fluorescent quantification kit miRNA Universal SYBR qPCR Master Mix for novzan, the detection system being shown in table 1:
TABLE 1 fluorescent quantitative PCR reaction System
The PCR program settings are shown in table 2:
TABLE 2 fluorescent quantitative PCR procedure
Detecting fluorescence signals of miR395b in different periods of lotus roots, wherein the intensity of the obtained fluorescence signals passes through 2 －△△ C T Analysis by this method gave the relative expression levels of miR395b, as shown in FIG. 2.
As can be seen from fig. 2, the amplification effect of miR395b is better in the initial stage of expanding lotus root-shaped stems, which indicates that miR395b plays an important role in the beginning of the initial stage.
Example 3
The present example was used to clone, verify and recover the target miRNA:
cloning of miR395 b: according to the sequence of miR395b gene, primer pairs (miR 395bF and miR395 bR) are designed, miR395bF and miR395bR are respectively shown as SEQ ID No.2 and SEQ ID No.3, and the lotus root cDNA prepared in example 1 is used as a template for PCR amplification of miR395b gene.
miR395bF:5'-GACTCTGCAGGTCGAATCATCAGGTGTCCCCTAGA-3'；miR395bR:5'-GATCTCTAGAGTCGAATAAAATCCAGCGGCCC-3'。
The PCR amplified product was subjected to 1.2% agarose gel electrophoresis, and a band of about 136bp was recovered and purified using a general agarose gel DNA recovery kit (Tiangen Biochemical Co., ltd. DP 209).
The resulting strip is shown in FIG. 3.
As shown in FIG. 3, miR395bF and miR395bR have obvious effect on cloning of target genes, the concentration of the strip distribution is located at the same position, the brightness is high, namely, the purity of the target genes is high and the obtained target genes are large.
Example 4
The embodiment is used for transferring a target gene to prepare a transgenic tobacco plant:
obtaining transgenic tobacco plants: cloning the full-length sequence of miR395b gene, connecting a Pcammia 2300 over-expression vector (a CaMV promoter sequence with 35S on the vector and a kanamycin selection marker gene KanR) and transforming an agrobacterium strain GV3101. Selecting a Nicotiana benthamiana plant which is pre-cultured in a callus induction culture medium for about 20 days, and placing the Nicotiana benthamiana plant in a conical flask containing agrobacterium tumefaciens liquid for shake co-culture for about 10min; the bacterial liquid is absorbed by sterile filter paper, the callus is placed in a callus induction culture medium to be cultivated for about 2 days in darkness (28 ℃), and after 3 times of flushing by sterile water, the callus is transferred to the callus induction culture medium containing 50mg/L kanamycin (Kan) for continuous cultivation; transferring the green callus with larger induction growth into a callus differentiation medium containing 50mg/LKan for culture; after a few days, the obtained adventitious buds are transferred into an MS culture medium containing 50mg/LKan and 1mg/mLNAA for rooting; when tobacco seedlings grow out, dividing the tobacco seedlings into miR395b OE-1-10 according to the strain, extracting DNA and RNA for transgenic identification to obtain positive seedlings, and dividing the obtained positive seedlings into the following according to different strains: miR395b OE-1, miR395b OE-6, miR395b OE-7, as shown in FIG. 4.
As can be seen from FIG. 4, a tobacco plant containing the target gene was produced.
Example 5
This example was used to verify the performance of transgenic plants:
starch content determination of transgenic plants: the transgenic plants and the control plants (tobacco leaves) are sampled and then deactivated for 1h at the temperature of 105 ℃ in an oven, then dried to constant weight at the constant temperature of 60 ℃, crushed and dried, and then the powder is collected for standby. The soluble sugar in the sample can be separated from the starch by using 80% ethanol, the starch is further decomposed into glucose by an acid hydrolysis method, and the total starch content of the lotus roots can be calculated by measuring the glucose content by using an anthrone colorimetric method. The specific method can be carried out by referring to the specification (BC 0700) of a starch content detection kit of Soy Co. The detection of amylopectin content was performed using the property of amylopectin and iodine to form a purple complex. The soluble sugar and starch in the sample are separated by ethanol, and iodine is used for reacting with the soluble sugar and starch to obtain the amylopectin content. Specific methods of detection are described in the Soy Corp amylopectin content detection kit (BC 4270).
The measured data are shown in fig. 5.
As can be seen from fig. 5, the total starch and amylopectin content of the transgenic plants was significantly reduced compared to the control group. At the same time, the amylose content is reduced to a lesser extent, the direct count ratio is increased to a certain extent, and the maximum can be increased from 0.6 to 0.78 of the comparative example.
Example 6
This example was used to perform starch chain length observations of transgenic plants:
and (3) observing the chain length of the starch of the transgenic plant: the transgenic material and the control material with the same growth vigor are collected, and the samples are collected and put into a dryer for storage for standby. Starch chain length was obtained by company detection.
As can be seen from fig. 6, the starch short chain in transgenic tobacco over-expressing miR395b is significantly lower than in burley, suggesting that miR395b can reduce starch accumulation by reducing the short chain length of transgenic plant starch.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (5)
1. An application of miRNA related to lotus root-like stem starch synthesis, which is characterized in that: comprising the steps of (a) a step of,
selecting plant material: extracting RNA of lotus root tubers in the mature period, namely miRNA;
cloning, acceptance and recovery of mirnas: designing a primer pair for miRNA, amplifying, electrophoresis and recovering;
transferring target genes and preparing transgenic plants: transforming the target gene into microorganism and infecting plant, and identifying to obtain positive seedling;
expression and detection: carrying out starch expression on the positive seedlings and detecting the starch expression quantity;
the gene sequence of the miRNA is shown as a Seq ID No.1, and the plant is tobacco callus which is infected and cultivated by callus induction;
expression of mirnas reduced the content of amylopectin and amylose.
2. The use according to claim 1, characterized in that: the miRNA has the length of 135bp.
3. Use according to claim 1 or 2, characterized in that: the expression of miRNA decreased amylopectin content by an extent greater than amylose.
4. The use according to claim 1, characterized in that: in the cloning, acceptance and recovery of miRNA, the primer pairs are as follows:
miR395bF:5'-GACTCTGCAGGTCGAATCATCAGGTGTCCCCTAGA-3'；
miR395bR:5'-GATCTCTAGAGTCGAATAAAATCCAGCGGCCC-3'。
5. the use according to claim 1, characterized in that: the microorganism is an agrobacterium strain GV3101 in the transgenic plant obtained by transferring the target gene.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101076592A (en) * | 2004-10-12 | 2007-11-21 | 洛克菲勒大学 | Micro rna |
| CN110904101A (en) * | 2018-09-14 | 2020-03-24 | 华中农业大学 | miR395 gene and regulatory site and application thereof |
| CN112980989A (en) * | 2021-03-24 | 2021-06-18 | 南京林业大学 | MiRNA fluorescent quantitative reference gene of different tissues of cryptomeria fortunei as well as primer and application thereof |
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| WO2014064704A2 (en) * | 2012-10-28 | 2014-05-01 | A.B. Seeds Ltd. | Transgenic plants with modified sugar content and methods of generating same |
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Patent Citations (3)
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| CN101076592A (en) * | 2004-10-12 | 2007-11-21 | 洛克菲勒大学 | Micro rna |
| CN110904101A (en) * | 2018-09-14 | 2020-03-24 | 华中农业大学 | miR395 gene and regulatory site and application thereof |
| CN112980989A (en) * | 2021-03-24 | 2021-06-18 | 南京林业大学 | MiRNA fluorescent quantitative reference gene of different tissues of cryptomeria fortunei as well as primer and application thereof |
Non-Patent Citations (2)
| Title |
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| Genome-Wide Analysis of MicroRNAs in Sacred Lotus, Nelumbo nucifera (Gaertn);Yun Zheng等;Tropical Plant Biol.;20130727;第6卷;117-130 * |
| Integrated mRNA and Small RNA Sequencing Reveals a microRNA Regulatory Network Associated with Starch Biosynthesis in Lotus (Nelumbo nucifera Gaertn.) Rhizomes;Yamei Zhu等;International Journal of Molecular Sciences;20220709;第23卷;1-14 * |
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