CN113444741B - Application of expression lowering Bna-miR168a in improvement of rape traits - Google Patents

Abstract

The invention discloses application of expression lowering Bna-miR168a in rape trait improvement. The invention discloses Bna-miR168a and rape seed yield, biological yield, seed-fruit ratio, harvest index, silique number and secondary effective branch number are closely related in shape, and the expression quantity of down-regulated Bna-miR168a can increase the seed yield, biological yield, seed-fruit ratio, harvest index, silique number, secondary effective branch number and the like of rape. Therefore, materials and methods for Bna-miR168a or regulating Bna-miR168a expression can be applied to realize improvement of plant varieties.

Description

Application of expression lowering Bna-miR168a in improvement of rape traits

Technical Field

The invention relates to the field of plant genetic engineering, in particular to application of expression lowering Bna-miR168a in improvement of rape traits.

Background

As one of three major oil crops in the world, rape is the oil crop with the largest planting area in China. However, as an economic crop mainly harvesting seeds, compared with crops with a harvest index of more than 50% such as rice and peanuts, the harvest index of the rape cultivar is only 20-30%, so that the improvement of the harvest index of the rape is particularly important for improving the yield of the rape. Among the three factors of 'source, stream and pool' affecting the harvest index, the rape has sufficient source and pool, so that the research on the transportation and distribution of stream, namely photosynthetic assimilate, becomes a breakthrough for improving the harvest index of the rape. The photosynthesis of rape in the green pod stage can provide more than 50% of photosynthetic products for the yield of seeds, the activity of sucrose phosphate synthase and the like in the carob peels influences the conversion of starch and sugar, the intensity of material transmission from the carob peels to the seeds is changed, and the high harvest index character is finally formed in the kernel-fruit ratio.

In the early stage research, the kernel-fruit ratio (the ratio of the dry weight of rape seeds to the dry weight of the siliques) and the thousand kernel weight (the dry weight of each thousand rape seeds) are used as target characters reflecting the high and low conversion efficiency of the photosynthetic products of the siliques, relevance analysis is carried out on the kernel-fruit ratio and the thousand kernel weight of 588 parts of heavy sequencing group materials for three years continuously, 12 candidate genes and 9 candidate differential expression miRNAs which are possibly related to the high-efficiency transportation of the photosynthetic products of the siliques are screened out in combination with the transcriptome of an extreme material and the small RNA sequencing analysis result, wherein bna-miR168a has extremely obvious difference in the extreme material, the specificity of miR168 and the complexity of the transportation process of the photosynthetic products are comprehensively considered, and bna-mi168a with a large possible regulation range is finally selected as a main candidate miRNA for functional research. At present, reports about miRNA mainly focus on model plants such as Arabidopsis, Chinese littleleaf poplar, rice and the like, and researches on Brassica napus are few, so that researches on the regulation and control mechanism of miRNA in the growth and development process of Brassica napus, especially researches on complex quantitative characters in Brassica napus, have very important significance on the genetic improvement and breeding of Brassica napus.

Disclosure of Invention

In view of the above, one of the purposes of the present invention is to provide an application of expression lowering Bna-miR168a in rape trait improvement; the second purpose of the invention is to provide a method for improving the yield of rape seeds.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of the expression of Bna-miR168a in the improvement of rape traits through down-regulation is characterized in that: the improved rape traits are as follows: the yield of the seeds is improved; the biological yield is improved; the grain-fruit ratio is improved; the harvest index is improved; increasing the quantity of siliques; the primary effective branch height is reduced; the number of secondary effective branches is increased.

In the invention, preferably, the expression of the down-regulated Bna-miR168a is a silencer of Bna-miR168a constructed based on a target mixed technology, and the expression of a Bna-miR168a mature sequence is specifically down-regulated.

Preferably, in the invention, the rape is cabbage type rape.

2. A method for improving the yield of rape seeds, which is characterized by comprising the following steps: the expression of the mature sequence of Bna-miR168a in rape is down-regulated.

In the invention, preferably, the expression of the Bna-miR168a mature sequence in the down-regulated rape is a silencer of Bna-miR168a constructed on the basis of target mimicry technology.

Preferably, the construction of the Bna-miR168a silencer based on the target mixing technology comprises the following steps:

(1) placing a mimicry Bna-miR168a sequence in an IPS1 gene sequence to obtain an MIM168a sequence segment;

(2) transferring the MIM168a sequence fragment into rape to obtain transgenic rape with improved characteristics.

In the invention, preferably, in the step (1), the MIM168a sequence is shown in SQE ID NO. 11.

Preferably, in the invention, the rape is cabbage type rape.

The invention has the beneficial effects that: compared with a receptor ZS11, the interference expression bna-miR168a in the brassica napus has the advantages that the interference expression plants grow vigorously, the plants are more in branches, the quantity of single siliques is more, and the characters such as biological yield, grain-fruit ratio, harvest index and the like are obviously higher. Meanwhile, three members of the BnAGO1 are target genes of bna-miR168a, the expression level of the BnAGO1 is negatively regulated by bna-miR168a, and the expression levels of bna-miR403, bna-miR164A, bna-miR156b and bna-miR396a are also regulated by bna-miR168a to different degrees. bna-miR168a can affect the combination of AGO1 and other miRNA to form an RNA silencing induction complex (RISC) through targeted negative control of AGO1, and the change of the bnamiR168a-AGO1 mediates the expression amounts of the bnamiR156b-SPL, the bnamiR396a-GRF and other bnamiRNAs-Targets so as to affect the characters of the plant such as the flowering phase, the branch number and the grain yield, and finally affect the yield characters of the plant such as the grain-fruit ratio and the harvest index.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a PCR check of bna-miR168a target fragment (A) and a bacterial suspension test (B) after bna-miR168a is ligated to the entry vector pENTR-D-TOPO;

FIG. 2 is a PCR detection map (A) of two target fragments of AtIPS 1; map (B) is detected after directional modification of an arabidopsis AtIPS1 target fragment; FIG. C shows the bacterial suspension after MIM168a was ligated to the entry vector pENTR-D-TOPO;

FIG. 3 is a schematic diagram of a fragment of overlapping PCR to construct MIM168 a;

FIG. 4 is a PCR detection chart of bacterial liquid obtained after BnamiR168a (A, B) and MIM168a (C, D) target fragments are connected to an expression vector pEarleyGate 101;

FIG. 5 is a screen of homozygous positive lines over-expressed bna-miR168a in Brassica napus (A, B are plants treated 6 days and 12 days after Basta spraying respectively; the left side of vertical line of ZS11 material is control without Basta spraying, and the right side of vertical line is treated with Basta spraying);

FIG. 6 shows the relative expression levels of bna-miR168a (A) and bna-MIR168a (B) in transgenic oilseed rape and ZS 11;

FIG. 7 shows the relative expression of the target gene BnAGO1 in transgenic rape plants and ZS 11;

FIG. 8 is a graph of the phenotype of Brassica napus ZS11 material and after 90 days of transplantation of lines (A) and individuals (B) overexpressing bna-miR168 a;

FIG. 9 is a scanning electron microscope photograph of flower organs of Zhongshuang 11 and bna-miR168a overexpression plants and detection of pollen viability under an optical microscope (ZS11 represents double 11 material in a receptor, OE168a represents an overexpression plant, A is a photograph under a body type microscope, B-E is a photograph under a scanning electron microscope, and E is a photograph for detecting pollen viability under an optical microscope. A is a flower organ with petals and sepals removed, B is pistil under 40 times, C is pistil stigma under 90 times, D is anther, E is pollen, and E is pollen after treatment of alexandric dye solution);

FIG. 10 is a graph of Brassica napus ZS11 material and phenotype and phenotypic statistics (C, D, E) after over-expression, interference-expression bna-miR168a strain (A) and individual (B) transplantation for 110 days;

FIG. 11 shows Brassica napus ZS11 material and phenotype of plants (A) expressing over-expression and interference expression bna-miR168a at harvest time and phenotypic statistics (B individual grain yield, C biological yield, D grain-to-fruit ratio, E harvest index);

FIG. 12 shows the relative expression levels of Bna-miR164A (A), Bna-miR403(B), Bna-miR156B (C) and Bna-miR396a (D) in bna-mi168a transgenic rape and ZS11 plants;

FIG. 13 is a screen for heterologous overexpression and interference in Arabidopsis thaliana showing homozygous positive lines bna-miR168 a;

FIG. 14 shows the relative expression levels of bna-miR168a (A) and bna-MIR168a (B) in transgenic plants and wild type;

FIG. 15 shows the relative expression level of the target gene AtAGO1 in transgenic Arabidopsis thaliana and wild type;

FIG. 16 shows the phenotype of transgenic and wild type Arabidopsis thaliana after 18(A), 23(B), and 26(C) days of seed planting;

FIG. 17 is a graph of the phenotype of transgenic and wild type plants 32(A) and 40(C) days after seed sowing, their bolting (B) and flowering time (D) statistics;

FIG. 18 is a phenotypic observation of transgenic and wild type Arabidopsis pod development (A), number of initial embryos (B) and number of fruit grains per horn (C);

FIG. 19 is a graph of silique length (A) and statistical analysis (B) of transgenic and wild type Arabidopsis plants at maturity;

FIG. 20 shows the phenotype of the transgenic plants at harvest time and wild type Arabidopsis (A) and its statistical analysis (B);

FIG. 21 shows the chlorophyll (A) and auxin (B) content in transgenic and wild type Arabidopsis leaves 34 days after seed-on-demand;

FIG. 22 is a scanning electron microscope photograph of heterologous overexpression bna-miR168a and various parts of a wild type Arabidopsis floral organ and detection of pollen viability under an optical microscope (WT represents a wild type Arabidopsis plant, OE168a represents a heterologous overexpression plant in Arabidopsis, A-E are photographs under a scanning electron microscope, C and D are photographs for pollen viability under an optical microscope, A is a floral organ from which petals and sepals are removed, B is pistil with a magnification of 100, C is pistil with a magnification of 200, D is anther, E is pollen, C and D are stigma and anther after treatment of alexander stain respectively);

FIG. 23 shows the relative expression amounts of bna-miR164A (A), bna-miR403(B), bna-miR156B (C) and bna-miR369a (D) in transgenic and wild type Arabidopsis plants.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 cloning of Brassica napus bna-miR168a Stem-Loop sequence

Searching stem-loop sequences of miRNA in miRBase (http:// www.mirbase.org /) database, designing primers BnamiR168a-F (SEQ ID NO.1) and BnamiR168a-R (SEQ ID NO.2) according to the stem-loop sequences (U in the stem-loop sequences is replaced by T), and adding four bases CACC at the 5' end of the front primer.

The method comprises the steps of taking mixed cDNA of roots, stems, leaves, flowers and buds of YC24 materials and horny pericarp and grains at different periods as a template, cloning a target fragment of bna-miR168a stem-loop sequence by using a primer BnamiR168a-F, BnamiR168a-R, obtaining a band with the size of about 450bp by agarose gel electrophoresis detection results, basically conforming to the size of the bna-miR168a stem-loop sequence in a miRBase (450bp) (figure 1, A), connecting the target band to pENTR-D-TOPO, transforming escherichia coli, carrying out PCR detection on monoclonal bacteria liquid (figure 1, B), detecting the primers M13-F + M13-R (SEQ ID No.3, SEQ ID No.4), and then sequencing to obtain the bna-miR168a fragment (SEQ ID No. 5).

Example 2 obtaining of Brassica napus MIM168a fragment

Therefore, the invention constructs the silencer of miR168a By directionally transforming the IPS1(Induced By Phosphate start 1) gene in the endogenous Target mix. The artificially modified mimicry Bna-miR168a sequence (MIM168a) plays a role in negative regulation and control of the expression of endogenous miR168a, and can be used for researching the influence on the crop phenotype after the interference expression of Bna-miR 168.

Primers AtIPS1-F (SEQ ID NO.6) and AtIPS1-R (SEQ ID NO.7) are designed according to two ends of a CDS sequence of an Arabidopsis AtIPS1 gene (At3G09922), CACC4 basic groups are added to the 5' end of a front primer, DNA of Arabidopsis is used as a template for cloning, and after PCR detection and sequencing are carried out on a gel recovery product, the AtIPS1 gene sequence (SEQ ID NO.8) is obtained.

Searching a bna-miR168a mature sequence in an online database by miRBase (http:// www.mirbase.org /), and designing primers BnMM 168a-F (SEQ ID NO.9) and BnMM 168a-R (SEQ ID NO.10) according to the mature sequence (U in the mature sequence is replaced by T); then, AtIPS1-F and BnIM 168a-R, BnIM 168a-F and AtIPS1-R are respectively used as two pairs of primers, AtIPS1 gene is used as an amplification template, two fragments are obtained after segmentation and cloning, and agarose gel electrophoresis results show that the sizes of the bands are basically consistent with the sizes of reference sequences (234bp and 268bp) (FIG. 2, A). Glue recovery is carried out; and finally, mixing the two gel recovery products obtained in the last step according to an equimolar amount to obtain a new template, carrying out overlapped PCR amplification by using AtIPS1-F and AtIPS1-R as primers to obtain an MIM168a fragment, and carrying out agarose gel electrophoresis to show that the size of the modified fragment is about 500bp and is basically consistent with that of a target fragment (478bp) (fig. 2, B). After the target band is connected to pENTR-D-TOPO, Escherichia coli is transformed, PCR detection of monoclonal bacteria liquid is carried out (figure 2, C), primers are M13-F + M13-R, and then sequencing is carried out to obtain a BnaMIM168a target fragment (SEQ ID NO. 11). A schematic diagram of the BnaMIM168a target fragment constructed by overlapping PCR is shown in FIG. 3.

Example 3, bna-construction of miR168a overexpression vector and interference expression vector

Respectively connecting two target fragments, namely bnamiR168a and BnaMIM168a to an expression vector pEarleyGate101 through LR reaction, respectively transforming escherichia coli, respectively carrying out PCR detection on F35S3ND (SEQ ID NO.12) + BnamiR168a-R, BnamiR168a-F + RPA3ND (SEQ ID NO.13) and F35S3ND + AtIPS1-R, AtIPS1-F + RPA3ND (figure 4) by using primer pairs, extracting a plasmid from a correctly sequenced bacterium solution, and transforming agrobacterium GV 3101.

Example 4 genetic transformation and Positive identification of Brassica napus

The bacterial liquid and plasmid with the completed construction and the correct sequencing are sent to Wuhanbo biotech company for the transfection experiment of the transgenic rape, and the transfection receptor is medium-double 11 material (ZS 11). Transferring the returned transgenic seedlings of the company into a Hoagland nutrient solution of 1/4 for culture, transferring the seedlings into a Hoagland nutrient solution of 1/2 for culture after one week, transferring the seedlings into a Hoagland nutrient solution of 1 time for culture about half a month, replacing the nutrient solution once a week until 4-5 cotyledons grow out, and transplanting the seedlings into soil for growth. And smearing Basta solution with concentration of 500mg/L on the rape leaves with normal growth, and marking with Marker pen. Observing leaf phenotype after one week, and preliminarily identifying plants with normal growth and dark green leaves as positive plants; the plants with aged and yellow leaves are negative plants. Carrying out DNA extraction on the leaves of the positive single-plant rape, and carrying out positive identification by adopting a PCR method; and selecting positive plants for further qRT-PCR verification, selecting single plants with the expression quantity of the transgenic plants reaching the level with obvious difference from ZS11 by taking ZS11 as a control, continuously planting, and harvesting the single plants in the harvest period.

Carrying out single plant harvest on plants which are positive in Basta screening, positive identification and qRT-PCR identification of the T0 generation, then carrying out seedbed seedling culture in 10 months according to the transgenic sowing requirement, spraying 500mg/L Basta solution for re-screening when 3-4 cotyledons grow out from the seedlings, and taking plants which grow normally and are tender green as positive plants; negative plants with yellow leaves and weak growth vigor are obtained. And transplanting the positive single plants in 11-month middle ten days. Until all seedlings are screened to be positive after Basta spraying, the homozygous positive transgenic lines are screened, and three positive individuals OE #1, OE #4 and OE #5 expressing bna-miR168a over-and three positive individuals MIM #6, MIM #7 and MIM #8 expressing bna-miR168 in an interference mode (namely BnaMIMR168 expressing) are obtained respectively.

Example 5 expression level of target Gene in transgenic Brassica napus

The screening results of homozygous positive lines of the over-expressed plants show that the proportion of the positive lines of the three over-expressed lines is close to 100% of that of the sown plants (figure 5), and the screening work of the homozygous positive lines is not carried out temporarily because the generation of the interference expression plants is lower; further qRT-PCR results showed that the expression levels of the mature sequences bna-miR168a of the over-expressed and interfering strains were 11, 39, 33 times and 0.25, 0.41, 0.51 times of ZS11, respectively, and both reached very significant levels of difference (fig. 6, a); in the over-expression strain, the expression level of the precursor bna-MIR168a is 3.3 times, 3.5 times and 3.3 times of that of the wild-type ZS11, and the difference is extremely remarkable, but the expression level of the precursor bna-MIR168a of 3 individuals interfering with the expression of bna-miR168a is not remarkably different from that of ZS11 (FIG. 6, B).

Example 6 target Gene detection of bna-miR168a in transgenic Brassica napus

Preliminary qRT-PCR verification is carried out on 6 target genes predicted by bna-miR168a in Brassica napus, and the result shows that only BnaC08g46720D, BnaA08g03260D, BnaA05g17460 and bna-miR168a have a negative regulation expression mode. The expression levels of BnaC08g46720D, BnaA08g03260D and BnaA05g17460 in the overexpression strain are respectively 0.75, 0.12 and 0.15 times, 0.69, 0.11 and 0.09 times and 0.71, 0.14 and 0.11 times of that of ZS 11; the expression quantity in the interference plant reaches 11 times, 16 times, 12 times, 15 times, 10 times and 7 times, 8 times and 5 times of the Zhongshuang 11 respectively (figure 7), and the 3 genes are just three members of target genes BnAGO1 of bna-miR168 a.

The results all prove that the screening accuracy of the homozygous positive strains which are overexpressed bna-miR168a in the brassica napus and the screening reliability of the positive strains which are interfered and expressed bna-miR168a can be used for carrying out next phenotype identification work.

Example 7 phenotypic statistics of interfering, overexpressing bna-miR168a positive lines in Brassica napus

The overall phenotype of the interference and over-expression bna-miR168a plants in the brassica napus shows that the over-expression bna-miR168a has an obvious growth inhibition effect on the brassica napus, and the interference expression plants have an obvious growth promotion effect. In the seedling stage, the growth vigor of the plants of the over-expression strain is weaker, when the ZS11 material reaches the full-bloom stage, the main inflorescence of the plants of the over-expression strain starts to bloom, and the flowering stage is about 15 days later than that of the ZS11 material (figure 8). And the preliminary observation of the interference expression shows that the florescence is earlier than that of the ZS11 material, and the whole seedling stage grows vigorously.

In the reproductive growth stage, the rape interfering expression is vigorous in growth, but the over-expressed rape line generally has abortion phenomenon of abnormal pod development. The ZS11 material and the flower organs of the over-expression line plants were divided into two stages: the bud without exposed petals is the first stage, the first flower which is opened under the bud is the second stage, and scanning electron microscope observation shows that the overexpression lines in the two stages respectively show a phenotype that the stigma is smaller (figure 9, B; figure 9, C), the cracking degree of the anther is obviously lower than that of ZS11 material (figure 9, D) in the same period, and the adhered pollen on the stigma is less (figure 9, B). Further detection of pollen viability of ZS11 and the over-expressed lines revealed that pollen viability was normal for both lines (fig. 9, e).

In the later stage of growth and development, the characters such as the branch number, the one-time effective branch height, the single plant silique number and the like of the transgenic plant are all obviously or very obviously different from those of ZS11 (figure 10). The branch numbers of the ZS11 material and over-expression and interference plants, particularly the number of the secondary effective branches are obviously different, the average ZS11 is 4, the three over-expression lines and the interference plants are respectively 1, 2, 19, 23 and 24, and the difference is extremely obvious (figure 10, C); the interfering plants have more branches and more dispersed plant types, the average height of the primary effective branches is 27cm, which is extremely obviously lower than 70cm of ZS11, the branches of the over-expression plants are fewer, and the primary effective branches are higher, which are 85cm on average (figure 10, D); the number of the siliques of the individual plants, which affects one of the three factors of yield, has a larger difference between the transgenic plants and the ZS11, the numbers of the siliques of the individual plants of the over-expression lines and the interference expression lines are 181, 206, 244 and 882, 920 and 956 respectively, and the difference reaches a remarkable or extremely remarkable level compared with 329 of ZS11 (FIG. 10, E).

In the harvest period, yield traits such as grain yield, biological yield, grain-fruit ratio and harvest index of the transgenic plants are all remarkably or extremely remarkably different from those of ZS11 (figure 11). The grain yield of the ZS11 material is 7.54g on average, the biological yields of the over-expression and interference expression strains are 5.56g, 5.64g, 5.2g and 29.3g, 31.1g and 39.9g respectively (FIG. 11, B), the biological yield of the ZS11 material is 42.78g on average, and the grain yield and the biological yield of the over-expression and interference expression strains are 45.24g, 46.1g, 42.98g and 120.5g, 119g and 146.6g respectively (FIG. 11, C), although the grain yield and the biological yield of the over-expression strains do not reach the difference significance compared with the ZS11, the grain yield and the biological yield of the interference expression strains are both significantly higher than that of the ZS11 material; the grain-fruit ratio traits reflecting the photosynthetic product transport efficiency are more significant in difference between the transgenics and the ZS11 material, compared with 39.99% grain-fruit ratio traits of the ZS11 material, the grain-fruit ratio traits of over-expression lines and interference-expression lines are respectively 27.03%, 24.18%, 25.49% and 59.09%, 64.24% and 62.45%, and the grain-fruit ratio traits reach the level of significant difference (figure 11, D); finally, the harvest index of the ZS11 material was 17.59%, the harvest index traits of the three overexpression lines were 12.46%, 12.04%, and 12.09%, respectively, which were all significantly lower than those of ZS11, and the harvest index traits of the three interfering expression lines were 24.31%, 26.13%, and 27.21%, respectively, which were all significantly higher than those of the ZS11 material (fig. 11, E).

Example 6 expression levels of other miRNAs in lines interfering and overexpressing bna-miR168a in Brassica napus

In order to verify whether the expression quantity of other miRNAs in transgenic strains of Brassica napus overexpression and interference expression bna-miR168a is influenced, the expression quantities of bna-miR164A, bna-miR403, bna-miR156b and bna-miR396a in positive strains of Brassica napus overexpression and interference expression bna-miR168a are detected, and the results show that bna-miR164A is down-regulated in three overexpression strains and is up-regulated in interference strains to reach 0.75, 0.52, 0.24 times and 1.6, 1.45 and 1.3 times of the ZS11 material respectively (FIG. 12, A). bna-miR403 was downregulated in both the overexpression and the interference strains, with expression amounts of 0.09, 0.25, 0.13-fold and 0.2, 0.35, 0.31-fold of ZS11, respectively (FIG. 12, B). bna-miR156b and bna-miR396a show the tendency of up-regulated expression in three over-expression strains, which are respectively 1.44, 1.86, 3.41 times and 4.81, 17.89 and 22 times of the ZS11 material, and are down-regulated expression in interference strains, which respectively reach 0.32, 0.52, 0.4 times and 0.06, 0.25 and 0.2 times of the ZS11 material (figure 12, C, figure 12 and D).

The results show that compared with a receptor ZS11, the overexpression and interference expression bna-miR168a in the brassica napus has the advantages that the overall flowering phase of the plants in an overexpression strain is delayed, the growth vigor is weak, secondary effective branches in a harvesting period are fewer, the number of single siliques is fewer, and the traits such as biological yield, grain-fruit ratio and harvesting index are all remarkably lower; the interference expression plants have vigorous growth, more branches, more siliques per plant, and significantly higher biological yield, grain-fruit ratio, harvest index and other properties. Meanwhile, three members of the BnAGO1 are target genes of bna-miR168a, the expression level of AGO1 is negatively regulated and controlled by bna-miR168a, and the expression levels of bna-miR403, bna-miR164A, bna-miR156b and bna-miR396a are also regulated and controlled by bna-miR168a to different degrees. bna-miR168a can affect the combination of AGO1 and other miRNA to form an RNA silencing induction complex (RISC) through targeted negative control of AGO1, and the change of the bnamiR168a-AGO1 mediates the expression amounts of the bnamiR156b-SPL, the bnamiR396a-GRF and other bnamiRNAs-Targets so as to affect the characters of the plant such as the flowering phase, the branch number and the grain yield, and finally affect the yield characters of the plant such as the grain-fruit ratio and the harvest index.

Comparative example:

genetic transformation and positive identification of arabidopsis thaliana bna-miR168a and BnaMIMR168

Carrying out a dip-dyeing experiment on the bacterial liquid of bna-miR168a and BnaMIMR168 positive recombinant expression vectors, mixing and harvesting seeds after maturation, dibbling the seeds in a culture medium containing Basta after disinfection, numbering positive plants with normal growth and green leaves, and transplanting the positive plants into soil. After 4-5 cotyledons grow out from arabidopsis thaliana, cutting off the leaves to extract DNA, and carrying out positive identification to obtain four positive individuals OE #10, OE #11, OE #12 and OE #14 in arabidopsis thaliana heterologous overexpression bna-miR168a and four positive individuals MIM #1, MIM #2, MIM #3 and MIM #5 in arabidopsis thaliana heterologous interference expression bna-miR168 a.

Acquisition and phenotypic identification of homozygous positive lines for heterologous expression of bna-miR168a in Arabidopsis thaliana

Through Basta screening, positive identification and qRT-PCR re-screening of early-stage multi-generation positive strains, four homozygous positive strains with heterologous overexpression bna-miR168a and four positive strains with interference expression are finally obtained from Arabidopsis, and the results show that the proportion of the positive strains of the eight strains is close to 100% of that of the seeds for dibbling (figure 13).

Further qRT-PCR results showed that the expression levels of the mature sequences bna-miR168a of the four over-and interference-expressing strains were 97, 242, 89, 244-fold and 0.35, 0.62, 0.06, 0.59-fold, respectively, of the wild type (fig. 14, a). The expression level of precursor bna-MIR168a in the over-expressed strain was 42393, 58113, 37998 and 83763 times that of the wild type, respectively, but the expression level of the precursor of the interfering strain did not tend to be stable as compared with the wild type (FIG. 14, B), probably because the interfering expression vector was constructed to act directly on the mature sequence without passing through the precursor.

Preliminary qRT-PCR verification is carried out on 4 target genes of bna-miR168a predicted in Arabidopsis, and the result shows that only the expression quantity of AtAGO1 and bna-miR168a have a negative regulation mode. The expression level of AtAGO1 in the overexpression and interference expression lines was 0.68, 0.60, 0.53, 0.37 fold and 6, 11, 14, 7 fold, respectively, that of the wild type (fig. 15). The results all prove the accuracy of screening the homozygous positive lines for heterologous expression bna-miR168a in Arabidopsis, and the next phenotype identification work can be carried out.

Phenotype statistical analysis is carried out on wild type and transgenic arabidopsis plants, and the results show that the growth of the over-expression strain is obviously inhibited, the yield is obviously reduced, the flowering phase of the plant of the interference strain is early, the number of branches is large, and the yield is improved. Specific phenotypic characteristics are as follows: at the seedling stage of vegetative growth, the over-expression strain shows weak growth, late growth, small and jagged leaves and obvious characteristics of an interference strain compared with wild arabidopsis thaliana, namely quick growth and large leaf (fig. 16).

In the process of converting nutrient growth into reproductive growth, bolting and flowering time of plants are obviously different, bolting and flowering time of wild plants is averagely 17 days and 32 days, bolting and flowering time of interfering strains are obviously earlier and are respectively 25 days, 24 days, 27 days, 30 days, 28 days and 29 days; the bolting and flowering time of the over-expressed strain was 32, 36, 33 days and 35, 38, 36 days, respectively, all significantly later (fig. 17).

In the reproductive growth stage, the interfering plants have no obvious difference compared with wild arabidopsis thaliana, but the over-expressed plants show extremely obvious silique abortion (fig. 18, a). Statistics of the number of initial embryos showed: no significant difference in the number of initial embryos was observed between the over-expressed and the interfering lines compared to the wild type (fig. 18, B), but there was a large difference in the number of seeds per silique at harvest time, the average number of seeds per silique in the over-expressed plants was 14, and a significant difference was observed between the over-expressed and the wild type (fig. 18, C), and the length of siliques in the interfering lines was not significantly different from the wild type, but the lengths of siliques in the four over-expressed lines were 0.9cm, 1cm, 0.8cm and 0.9cm, respectively, and a significant difference was observed between the wild type and 1.5cm (fig. 19).

After the maturity period, compared with the transgenic plants, the wild plants have significant differences in plant height, effective secondary branch number, single plant silique number, silique length, silique number per silique, grain yield, grain-fruit ratio, harvest index and other properties. The effective secondary branching of wild type plants was 4.9, and the overexpression and interference lines were 7.8, 9, 9.1 and 17.8, 13.8, 14, 11, respectively, all reaching a very significant level of difference (FIG. 20, A; FIG. 20, B-a). The average number of siliques of the wild type Arabidopsis plant is 162, and the overexpression and interference strains are 232, 230, 225, 234 and 327, 262, 255 and 232 respectively, which reach the level of extremely obvious difference (FIG. 20, A; FIG. 20, B-B). The biological yield of wild-type plants averaged 0.54g, with only 0.63g of the OE #11 strain out of the four overexpression lines reaching a differential significance with wild-type, while the other three lines, except MIM #5, reached a differential significance of 0.77g, 0.65g and 0.64g, respectively, out of the four interfering lines (fig. 20, a; fig. 20, B-c). In the aspect of single plant grain yield, the over-expression and interference strains are respectively 0.09g, 0.08g, 0.07g, 0.08g, 0.25g, 0.21g, 0.20g and 0.15g, while the wild single plant grain yield is averagely 0.13g, which achieves the level of significant difference or extremely significant difference (figure 20, A; figure 20, B-d). In terms of thousand seed weight, the interference strain and the wild type strain are about 0.0130g and have no significant difference, but the overexpression strains are respectively 0.01g, 0.007g, 0.008g and 0.009g, and all reach significant difference levels compared with the wild type (figure 20, A; figure 20, B-e). The kernel-to-fruit ratio of the wild type plants averaged 59%, with over-expression and interference lines 43.98%, 30.13%, 31.75%, 36.8% and 67.56%, 65.65%, 64.73%, 61.66%, respectively, and also 7 lines, with the exception of MIM #5 line, were very significantly different from the wild type (FIG. 20, A; FIG. 20, B-f). The harvest index traits of both the final over-expressed and interference lines reached very significant levels of difference compared to 24.85% of wild type, 18.26%, 12.90%, 12.9%, 15.74 and 32.9%, 33.13%, 30.63%, 31.03%, respectively (FIG. 20, A; FIG. 20, B-g).

By combining the above, it can be seen that after bna-miR168a is expressed by heterologous overexpression, the growth phenomenon of the Arabidopsis plant is obviously inhibited, the yield-related traits are all obviously reduced, and after bna-miR168a is expressed by heterologous interference in Arabidopsis, the plant can show an extremely obvious phenomenon beneficial to the growth and development of the plant from the seedling stage.

The phenomena of abnormal leaf development, obvious saw teeth and the like of a transgenic plant can be one of the reasons influencing the yield of the plant, and the contents of chlorophyll and auxin in the leaves after the transgenic and wild type arabidopsis thaliana are sowed for 34 days are determined, so that chlorophyll a has no difference between the transgenic and wild type plants, chlorophyll b is lower in an overexpression and an interference expression strain than in the wild type, the total chlorophyll amount of the interference expression strain is 1.67mg/g, the difference significance is not achieved with 1.74mg/g of the wild type, the average total chloroplast amount of the overexpression strain is 1.59mg/g, and the difference level is achieved with the wild type plant (figure 21, A); compared with wild arabidopsis, IAA is significantly lower in both overexpression and interference expression strains, the IBA content in the overexpression plant and the wild arabidopsis is not different, the IBA content in the interference plant is significantly higher than that in the wild arabidopsis, the total auxin amount in the wild type is 3.8ng/g, the total auxin amount in the overexpression and interference expression strains is 3.1ng/g and 2.9ng/g respectively, and the total auxin amount in the overexpression and interference expression strains is significantly lower than that in the wild type arabidopsis (fig. 21, B). We speculate that low chlorophyll and low auxin content in leaves of over-expressing plants directly affects the growth and development of leaves, whereas low auxin content in interfering expressing plants compared to wild type plants may be due to the difference in growth rate between interfering plants and wild type arabidopsis thaliana.

To explore the severe abortion phenomenon in the reproductive growth stage of over-expressed plants, we mainly divided the reproductive growth stage in the prophase of arabidopsis pods into three stages: early Stage of still unexposed buds (fig. 22, Stage 1), mid-Stage of fully extended four petals (fig. 22, Stage 2), and late Stage of sepal and petal abscission (fig. 22, Stage 3). Scanning electron microscopy of pistils, stamens, stigma, anthers and pollen grains in three stages of over-expressed and wild-type plants, respectively, revealed that anthers of over-expressed plants produced pollen (FIG. 22, D; FIG. 22, E), but the stage of anthers of over-expressed plants produced pollen was delayed compared to wild-type Arabidopsis, and not synchronized with gynoecium stigma growth (FIG. 22, A; FIG. 22, B). In addition, we also observed that the stigma of the over-expressed plants had a significantly enlarged characteristic compared to that of the wild type, and the epidermal cells on the stigma extended into villi bodies longer (fig. 22, C). In the middle and later stages of Arabidopsis pod development, more pollen grains are adhered to the stigma of the wild type plant, but the adhered pollen grains of the over-expression plant stigma are less (FIG. 22, B; FIG. 22, C).

To further clarify the problem of pollen grain activity between wild type and over-expressed plants, we performed pollen activity assays on organs such as anthers, stigma, etc. at three stages. The results showed that pollen viability of the over-expressed plants was normal (fig. 22, d). Dynamic phase observations still demonstrated that the anthers of the over-expressed plants shed pollen grains with a lag time and that less pollen grains adhered to the stigma were viable throughout pod development (FIG. 22, c).

In view of the specificity of targeting AGO1 by miR168, the expression amounts of bna-miR164A, bna-miR403, bna-miR156b and bna-miR396a are detected in a positive strain heterologously expressing bna-miR168a, and the results show that: bna-miR164A, bna-miR403, bna-miR156b and bna-miR396a all present different degrees of down-regulation expression trends, and the expression amounts of the down-regulation expression trends respectively reach 0.44, 0.49, 0.25 and 0.46 times, 0.18, 0.17, 0.04 and 0.15 times, 0.74, 0.65, 0.33 and 0.76 times, 0.69, 0.60, 0.15 and 0.47 times of wild-type plants; in four interference expression strains, bna-miR164A, bna-miR403, bna-miR156b and bna-miR396a show up-regulated expression trends in different degrees, the expression amounts of the expression trends respectively reach 1.19, 1.73, 1.42 and 1.82 times, 1.18, 2.60, 3 and 2.33 times, 1.36, 3.3, 6.16 and 5 times, 1.73, 3.18, 7.16 and 6.37 times of wild-type plants (figure 23), and the differences or extremely significant levels are achieved.

In conclusion, by phenotype observation of transgenic arabidopsis among multiple generations, it is found that plants of a heterologous overexpression and interference expression bna-miR168a positive homozygous strain in arabidopsis have relatively stable and very obvious phenotypic traits different from those of wild arabidopsis, the overexpression plants show reverse inhibition effect in the whole growth and development stage, and on the contrary, the growth and development of the interference strain plants have obvious promotion effect.

In this study, the transgenic phenotype of bna-miR168a in Arabidopsis plants: the bolting time of the over-expression strain is about 8 days later than that of the wild type, the flowering time is delayed by nearly 5 days, but compared with the wild type strain, the interference strain has the characteristics of obvious early bolting and early flowering; the seed test data of the yield-related characters of the plants in the mature period show that compared with wild type plants, the over-expressed plant types are dispersed, the grain-fruit ratio and the harvest index in the harvest period are not enough than 3/5 of the wild type plants, and particularly, the grain number and thousand seed weight of each horn of three factors influencing the plant yield are obviously reduced, so that the grain yield of the over-expressed plants is reduced by 38%; the effective secondary branching number of the interference strain is 2-3 times of that of the wild type, the grain-fruit ratio and the harvest index are respectively improved by 10% and 30%, and the yield of single plant grains is improved by 55% compared with the wild type.

Molecular level detection shows that AtAGO1 has negative expression trend with bna-miR168a in over-expressed and interference plants. In view of the fact that all miRNAs can be involved in regulating and controlling the expression of a target gene after being combined with a RISC complex, and miR168 is a special miRNA for regulating and controlling a core element AGO1 of the RISC complex, we preliminarily detect that the expression amounts of bna-miR403, Bna-miR164A, Bna-miR156b and Bna-miR396a in a transgenic plant of bna-miR168a are regulated and controlled by bna-168 miR168a to different degrees, which proves that miR168 and miR403 can jointly regulate the expression amounts of AGO1 and AGO2 by foreigners, and the change of a regulation signal of 'miR 168-AGO 1' formed by Osa-miR168 and a target gene AGO1 can cause the change of a plurality of other 'miRNA-Targets' regulation signals.

The research also finds that the over-expression plants in the seedling stage show extremely obvious characteristics of leaf development retardation, serration and the like, and the remarkable silique abortion phenomenon appears in the reproductive growth stage. Aiming at the abnormal phenomenon of the leaves, the detection of auxin and chlorophyll of transgenic and wild type arabidopsis leaves 35 days after the seeds are dibbled discovers that the total amount of chlorophyll of an interference strain is not different from that of the wild type, but the chlorophyll content of an overexpression strain reaches a very obviously lower level; compared with wild type, the total amount of auxin of over-expressed and interfering strains is obviously reduced, and the change of the regulation signal of miR168-AGO1 is presumed to cause the change of the expression quantity of miRNA involved in auxin regulation such as miR164, miR160, miR167 and the like. Aiming at the condition that the pod of an over-expression plant is seriously aborted, researches show that the number of the starting embryos of the pod between a transgenic plant and wild arabidopsis thaliana is not different, the pollen activity detection also shows that the pollen activity of the over-expression plant is normal, but the scanning electron microscope observation on the whole dynamic development process of anthers, pollen, stigma and other flower organs shows that the time for the anthers of the over-expression plant to emit pollen is lagged, so that the stigma overgrowth is caused, the time for pollen adhesion is missed, and the change of a regulation signal of miR168-AGO1 is presumed to influence miRNAs such as miR164 and the like, so that a targeted mediated transcription factor regulates and controls genes such as CUC and the like, and finally the quantity and the development of the flower organs of the plant are greatly influenced.

By combining the above, it is presumed that AtAGO1 in Arabidopsis is a true target gene of bna-miR168a, and that expression of AtAGO1 is negatively regulated by bna-miR168a, and other miRNAs such as bna-miR403, bna-miR164A, bna-miR156b, bna-miR396a and the like are combined with AtAGO1 to form a RISC complex, so that the regulation effect on the target genes of other miRNAs is further achieved, and the growth, development and yield traits of plants are finally influenced. The zigzag leaf character of an arabidopsis heterologous overexpression bna-miR168a strain is likely to be the change of 'bnamiR 168a-AGO 1', influences the combination of other 'bnamiRNA-AGO 1', plays a role in regulating and controlling genes for downstream regulation and control of auxin transport, enables auxin in leaves to be unevenly distributed, has obvious zigzag, influences the chlorophyll content in the leaves, and causes the final plant yield to form obvious difference. On the basis that the number of initial embryos is not remarkably different and the activity of pollen is not influenced, an obvious abortion phenomenon appears in an over-expression strain, and we preliminarily conclude that the sugar transport gene in the development process of pollen and other floral organs is down-regulated to cause insufficient nutrient supply, so that the anther, pollen and other organs and stigma are asynchronously grown, the pollen with less adhesion on the stigma is caused, and the serious abortion phenomenon finally causes the total yield of plants to be reduced.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

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Claims (8)

1. Down-regulating expressionBna-miR168a The application in improving the rape traits is characterized in that: the improved rape traits are as follows: the yield of the seeds is improved; the biological yield is improved; the grain-fruit ratio is improved; the harvest index is improved; increasing the quantity of siliques; the primary effective branch height is reduced; and the number of secondary effective branches is increased.

2. Use according to claim 1, characterized in that: the downregulation of expressionBna-miR168a Is constructed based on the targetimitry technologyBna-miR168a Specific down-regulation ofBna-miR168a Expression of the mature sequence.

3. Use according to any one of claims 1 to 2, characterized in that: the rape is cabbage type rape.

4. A method for improving the yield of rape seeds, which is characterized by comprising the following steps: down-regulation in oilseed rapeBna- miR168aExpression of the mature sequence.

5. The method of claim 4, wherein: said down-regulation in oilseed rapeBna-miR168a Expression of mature sequence is based on target mimicry technology constructionBna-miR168a The silencer of (1).

6. The method of claim 5, wherein the target-based mimicry technology is constructedBna-miR168a The silencer of (a) comprises:

(1) will mix the libraryBna-miR168a The sequence is placed in an IPS1 gene sequence to obtain an MIM168a sequence fragment;

(2) transferring the MIM168a sequence fragment into rape to obtain transgenic rape with improved characteristics.

7. The method of claim 6, wherein: in the step (1), the MIM168a sequence is shown as SQE ID NO. 11.

8. The method according to any one of claims 4 to 7, wherein: the rape is cabbage type rape.

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