Application of gene miR408 and UCL in cultivating high-yielding rice

A technology for transgenic rice and paddy rice, applied in the field of application in cultivating high-yielding rice

Active Publication Date: 2015-12-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in order to solve the contradiction between the ever-increasing population and the gradually decreasi...

Method used

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  • Application of gene miR408 and UCL in cultivating high-yielding rice
  • Application of gene miR408 and UCL in cultivating high-yielding rice
  • Application of gene miR408 and UCL in cultivating high-yielding rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1 rice total RNA extraction method

[0091] In the following examples of the present invention, the phenol-chloroform-isoamyl alcohol method is used to extract rice total RNA (including wild-type and various mutant rice), and the specific steps are as follows:

[0092] (1) Weigh 1g of rice sample and grind it into fine powder with liquid nitrogen, add 10mL RNAzol (100ml contains 47.2g of guanidine isothiocyanate, 2.5ml of 1M sodium citrate (pH7.0), 5ml of 10% sodium lauroyl anitrate) and 1mL sodium acetate (2M, pH4.0), continue grinding;

[0093] (2) Add 10mL of water-saturated phenol, 4mL of chloroform: isoamyl alcohol (49:1, volume ratio) mixture, mix well, transfer to a centrifuge tube, vortex for 1min, place on ice for 5 minutes, then 4°C, Centrifuge at 12000rpm for 15min;

[0094] (3) Take the supernatant after centrifugation, and add an equal volume of phenol:chloroform:isoamyl alcohol (50:49:1, volume ratio) mixture to the supernatant, vortex for 1...

Embodiment 2

[0102] Example 2 Construction of rice mutants overexpressing miR408

[0103] 1. Construction of MiR408 constitutive expression plasmid

[0104] (1) In this example, the vector pCAMBIA1390 (commercially available) was selected, and at the restriction site of the vector Hind III and Sal The CaMV35S promoter was inserted between I, which can be used as a constitutive promoter to control the expression of miR408 in the future. The experimental operation here adopts the routine technique in this field.

[0105] (2) Using the total RNA of the wild-type rice treatment sample extracted by the method in Example 1 as a template, the miR408 gene was cloned. The nucleotide sequence of the upstream primer F1 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO. .4, and add at both ends of the amplification product EcoRI with BglII enzyme cleavage site. For the PCR amplification reaction conditions, refer to the instructions of th...

Embodiment 3

[0126] Example 3 Construction of UCL-RNA Interference Rice Mutants

[0127] 1. Construction of UCL-RNA interference expression plasmid

[0128] (1) The RNA interference vector used in this example was provided by Mr. Yaoguang Liu of South China Agricultural University (the RNA interference vector was transformed from vectors pCAMBIA1305.2, pUC18-Pubi and pZEro-T).

[0129] (2) Using the total RNA of the wild-type rice treatment sample extracted by the method in Example 1 as a template, the UCL gene was cloned. The nucleotide sequence of the upstream primer F2 is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer R2 is shown in SEQ ID NO. .6, and add at both ends of the amplification product Himd III and Bamh Restriction site for I. For the PCR amplification reaction conditions, refer to the instructions of the PCR amplification reaction kit.

[0130] (3) After PCR, the amplified product was recovered and carried out Himd III and Bamh I doubl...

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Abstract

The invention discloses application of gene miR408 and UCL (uclacyanin) in cultivating high-yielding rice. By analyzing miRNA in charge of specific expression in rice grains and embryos, it is found that the gene miR408 has an important regulating effect in rice grain growth, and the function is achieved by lowering the target gene UCL of the gene miR408. According to the application, rice mutant strains over-expressing miR408 and UCL-RNA interference and UCL-CRISPR knockout are respectively constructed. The research shows that the three types of mutant strains all have the phenomenon of rapid growth, and the drooping phenomenon of rice ears at the maturation stage is more obvious compared with that of a wild type; in addition, the fruiting rate is increased, ears are larger in size, the number of grains per ear is much larger compared with the wild type, and the thousand grain weight and the yield are also remarkably increased. According to the application, a brand-new, simple and effective method is provided for cultivation of high-yielding rice and can be applied and popularized on a large scale; besides, the application will not pollute the environment, and the rice is safe to eat.

Description

technical field [0001] The invention belongs to the technical field of plant breeding. More specifically, it relates to the application of genes miR408 and UCL in breeding high-yielding rice. Background technique [0002] The food problem is one of several difficult problems facing the world today. Finding genes that affect yield-related traits in rice has long been a research focus for improving food production. So far, many genes affecting rice yield have been discovered, providing a theoretical basis for improving rice yield. [0003] miRNA (microRNA) is a group of non-coding single-stranded RNA widely present in multicellular organisms such as animals and plants. It can induce the degradation of the mRNA or inhibit the synthesis of the protein by binding to a specific site of the target gene mRNA. It leads to gene silencing at the transcriptional or post-transcriptional level, thereby participating in the regulation of gene expression. miRNAs are involved in almost a...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 张金平李权峰张玉婵于洋冯彦钊陈月琴
Owner SUN YAT SEN UNIV
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