WO2022257697A1 - Cll1 gene for regulating and controlling semi-dwarf plant type and leaf ratio of plant and use of leguminous orthologous gene of same - Google Patents

Abstract

The present invention belongs to the technical field of genes of plants. More specifically, provided in the present invention are a CLL1 gene for regulating and controlling the elongation of the lateral organ of a plant and the use thereof. According to the present invention, by means of regulating and controlling the CLL1 gene and a CLL1 protein encoded thereby, the blade/petiole ratio of leguminous plants and forage grass can be greatly increased, and meanwhile, the plant can present a semi-dwarf phenotype, so that the photosynthetic efficiency is improved, and the edible part of the forage grass is maximized.

Description

Application of the CLL1 gene and its orthologous genes in leguminous plants regulating the semi-dwarf plant type and leaf ratio technical field

The invention belongs to the field of plant gene technology, more specifically, the invention relates to the CLL1 gene and its orthologous gene for regulating the elongation of plant lateral organs and their applications.

Background technique

Mitogen-activated protein kinase (MAPK) is a serine/threonine protein kinase that is very conserved in eukaryotes and plays a role in many cellular activities, such as growth and proliferation, cell differentiation, cell motility or death. The MAPK cascade signal transduction is composed of three different levels of molecules. MAPK is activated by phosphorylation of MAPK kinase (MAPKK), and MAPKK is activated by phosphorylation of MAPKK kinase (MAPKKK). MAPKKK is activated by interacting with GTPase and/or other proteases, thereby linking MAPK with receptors on the cell surface and extracellular signals.

Stomata in Arabidopsis are a structure specialized for gas exchange with the external environment during the evolution of plants from aquatic to terrestrial. The stomatal complex is composed of a pair of guard cells and the micropores in the middle, through the opening and closing of the stomata, the exchange of CO ₂ and oxygen with the outside world is achieved. Studies have shown that the phosphokinase pathway (MAPK cascade pathway) may be involved in the early development of plant stomata. The developmental signal of the stomata is first transmitted by the EPF1/EPF2 short peptide, and is transmitted by the receptor kinase TOO MANY MOUTHS (TMM) on the cell membrane and the ER family. ERECTA(ER), Erecta-like1(ERL1) and ERL2 are delivered into cells, negatively regulated by YODA-MKK4/5-MPK3/6, and eventually phosphorylate SPEECHLESS(SPCH) in the nucleus, leading to its degradation and regulating the fate of the stomata. In addition to regulating stomatal development, the YODA-MKK4/5-MPK3/6 pathway is also involved in the morphogenesis of plant organs such as inflorescence, fruiting stalk, tap root and lateral root in Arabidopsis. Studies have shown that in this pathway, the loss of any first-level member will lead to the shortening of inflorescence and fruit stalk, and the formation of deformed inflorescence. In addition, Xu and Guo et al. proved genetically and biochemically that the YODA-MKK4-MAPK6 cascade pathway positively regulates rice seed size and panicle development, thereby directly determining rice yield.

On the other hand, other studies have shown that the MKK4/5-MPK3/6 pathway in Arabidopsis regulates the abnormal abscission of Arabidopsis floral organs. Shedding refers to the phenomenon that the infected or non-functional organs are separated from the mother body. Shedding can make the plants use their own resources more reasonably, so as to grow and develop better. Genetic evidence shows that INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is the upstream of this pathway, HAESA/HAESA-LIKE2 (HAE/HSL2) single protrusion has no phenotype, and double protrusion shows that the flower organ does not shed, indicating that the membrane signal of the shedding signal is Reception requires the presence of 2 proteins. The signal is further transmitted to the MAPK cascade pathway, and the signal is amplified step by step through multi-level phosphorylation. However, the direct upstream of MKK4/5 and the downstream of MPK3/6 that regulate floral organ abscission still need further study. The initiation of lateral roots, taking Arabidopsis as an example, can be simplified as the initiation of lateral root primordia, the formation of lateral root primordia, and the breakthrough of lateral roots through the epidermis. In the process of breaking through the epidermis, it passes through the inner cortex, the cortex and the epidermis successively. Kumpf et al. proved that the number of lateral roots decreased after the IDA-HAE/HSL2 mutation, mainly due to the abnormal degradation of pectin in the cell wall when the lateral roots broke through the three-layer tissue site, resulting in the abnormal development of lateral roots. At the same time, genetic and biochemical results showed that MKK4/5-MPK3/6 was located downstream of IDA-HAE/HSL2 to regulate the development of lateral roots, and cell wall degradation was mainly caused by PG ABSCISSION ZONE ARABIDOPSIS THALIANA (PGAZAT), PG LATERAL ROOT (PGLR) , XTH23/XYLOGLUCAN ENDOTRANSGLYCOSYLASE 6 (XTR6) and EXPANSIN 17 (EXP17) expression effects.

Existing research results show that the phosphokinase pathway is a very conserved pathway in plants. During plant growth, the phosphokinase pathway is widely involved in various aspects of plant growth and development. However, the function of the phosphokinase pathway in other species is not well understood. The molecular mechanisms behind morphogenesis of plant organs, especially petiole and rachis, are largely unknown.

Contents of the invention

In view of this, the present invention provides a CLL1 gene and its legume orthologous gene that positively regulate the elongation of the lateral organs of the plant, through the regulation of the CLL1 gene, the ratio of leaves/petioles is increased, and the stalk of the plant is adjusted at the same time. High, thereby improving the photosynthetic efficiency of crops and the utilization rate of pasture.

In a first aspect, the present invention provides a mutated Medicago truncatula CLL1 gene, compared with Medtr1g100250, the first intron of the mutated Medicago truncatula CLL1 gene is deleted, and the first intron sequence of the deletion is as SEQ ID NO.1 is shown. The nucleotide sequence of the mutated Medicago truncatula CLL1 gene is shown in SEQ ID NO.2, and the encoded CLL1 protein sequence is shown in SEQ ID NO.3.

In the second aspect, the present invention provides the use of the CLL1 protein described in the first aspect of the present invention in regulating the elongation of plant lateral organs, and the regulation is positive regulation.

In the third aspect, the present invention provides a kind of Medicago truncatula cll1 mutant, which is characterized in that, compared with the wild type, the cll1 mutant contains the mutated Medicago truncatula CLL1 gene described in the first aspect of the present invention, and its nucleus The nucleotide sequence is shown in SEQ ID NO.2.

In the fourth aspect, the present invention provides a method for improving plants, the method comprising down-regulating the expression of CLL1 gene, such as removing the first intron of the CLL1 gene or using RNA interference technology to reduce the expression of CLL1 gene.

In some embodiments, the fragment used for RNA interference is as shown in SEQ ID NO.4.

In the fifth aspect, the present invention provides a fusion expression vector, which includes the gene sequence to be expressed and the first intron of the CLL1 gene inserted and replaced.

In some embodiments, the gene sequence to be expressed in the fusion expression vector is shown in SEQ ID NO.5; the first intron sequence of the CLL1 gene is shown in SEQ ID NO.1.

In the sixth aspect, the present invention provides the legume orthologous protein of the CLL1 protein described in the second aspect, the amino acid sequence of the protein is shown in SEQ ID NO.6-20, and the legume plant includes: hawk Beans (Cicer arietinum), peas (Pisum sativum), soybeans (Glycine max), kidney beans (Phaseolus vulgaris), red beans (Vigna angularis), groundnuts (Arachis hypogaea), vines (Arachis duranensis), mung beans (Vigna radiata) Trifolium pratense, alfalfa (Medicago sativa), and lotus japonicus.

In the seventh aspect, the present invention provides a gene encoding a legume orthologous protein of the CLL1 protein, and the nucleotide sequence of the gene is shown in SEQ ID NO.21-35.

Beneficial effects of the present invention:

1) Provide a mutated CLL1 gene that positively regulates the elongation of lateral organs of plants, which can be used to regulate the growth and development of plants, especially to increase the ratio of leaves/petioles and make plants exhibit semi-dwarf phenotypes;

2) Provide the orthologous gene of CLL1 gene in other important leguminous crops and the application of its encoded protein in regulating the elongation of plant lateral organs;

3) Provide a method for improving plants by regulating the CLL1 gene and its legume orthologs, the method can greatly increase the ratio of leaves/petioles of legumes and pastures, while the plants present a semi-dwarf Phenotypes, thereby increasing crop yield and maximizing the edible parts (leaves) of forage grasses, providing a reference resource for molecular breeding.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those skilled in the art, other implementations can also be obtained according to these drawings without creative efforts.

Figure 1 shows the molecular cloning of CLL1; among them, Figure 1A shows that the CLL1 locus was first mapped to the long arm of chromosome 1, between the markers fgp2468 and 456-2, and further restricted to between fgp2468 and fgp2472 120kb region between; Figure 1B shows 17 candidate genes located in the 120kb region, the 13th gene is Medtr1g100250; Figure 1C shows the gene structure of CLL1 in wild-type and cll1 mutant plants, black boxes and The lines represent exons and introns respectively, the red line represents the first intron in CLL1, and the arrows above and below the gene structure represent different primers; Figure 1D shows the PCR of partial sequences of CLL1gDNA in wild-type and cll1 mutant plants Amplification; Figure 1E shows RT-PCR analysis of CLL1 transcripts in wild-type and cll1 mutants; Figure 1F shows the relative expression levels of CLL1 in wild-type and cll1 mutants, the MtGAPDH gene was used as an internal control; data The mean value (+SD) was taken, n=3 (Fig. 1F); * represents statistically significant difference by Student's t-test (*, P<0.05; **, P<0.01), the above experiments were repeated three times, and the results were similar.

Figure 2 shows that the first intron of CLL1 stimulates LUC activity; Figure 2A shows the LUC structure of the CLL1 promoter and the schematic diagram of the heterologous connection of the first intron of CLL1 behind Arabidopsis TRP1, and the hollow boxes are respectively Represents the promoter regions of CLL1 and TRP1, the red short box represents the first intron of CLL1, the gray box represents part of the coding region of AtTRP (the first intron of CLL1 is inserted in the coding region of AtTRP), and the black box represents LUC Gene, pCLL1::LUC is abbreviated as CLL1-p, and pCLL1-Intron::LUC is abbreviated as CLL1-p-intron1; Figure 2B and Figure 2D show the expression of CLL1-p-intron1 and AtTRP-p-intron1 in tobacco leaves Transactivation; Figure 2C and Figure 2E show the ratio of firefly luciferase (LUC) to Renilla luciferase (REN) activity; data are averaged (+SD), n=3 (Figure 2C and Figure 2E ); * represents statistically significant difference by Student's t-test (*, P<0.05; **, P<0.01), the above experiments were repeated three times, and the results were similar.

Figure 3 shows the petioles and rachis in transgenic lines with reduced CLL1 gene expression; wherein, Figure 3A shows mature compound leaves of representative wild-type, RNAi-1 and RNAi-2 transgenic lines (7 weeks) ; Figure 3B shows the SEM image of the petiole surface of the 7-week-old plant of wild type, RNAi-1 and RNAi-2; Figure 3C shows the 7-week-old plant of wild type, RNAi-1 and RNAi-2 plant SEM image of leaf axis surface in Section 4; Figure 3D and Figure 3E show the relative expression levels of CLL1 and Medtr8g098425 in wild-type, RNAi-1 and RNAi-2 plants, respectively, wherein the expression levels of CLL1 and Medtr8g098425 in wild-type Value is set to 1, and data are averaged (+SD), n=3 (Fig. 3D and Fig. 3E), MtGAPDH gene is used as internal control; Fig. 3F shows the petiole length of the mature compound leaf of 7 weeks old plant; Fig. 3G and Fig. 3H show the petiole epidermal cell length and the number of petiole cells of the mature compound leaves on the 4th node of the 7-week-old plant; Fig. 3I shows the leaf axis length of the mature compound leaf of the 7-week-old plant; Fig. 3J and Fig. 3K shows the length of the epidermis cell length and the number of cells in the axis of the mature compound leaf on the 4th node of the 7-week-old plant; the data are averaged (+SD), n≥9 (Fig. 3F, Fig. 3H, Fig. 3I and Fig. 3K), n≥28 (Fig. 3G and Fig. 3J); * represents statistically significant difference between wild type and RNAi-1 or RNAi-2 by Student's t-test (*, P<0.05; **, P<0.01), ns means not significant, and the scale bar is 2 cm (Fig. 3A) and 20 μm (Fig. 3B-Fig. 3C).

Fig. 4 shows the plant height (10 weeks) of the transgenic line that CLL1 gene expression reduces; Wherein, Fig. 4A shows the photograph of the plant height of wild type, RNAi-1 and RNAi-2 transgenic line; Fig. 4B shows The control figure of the plant height of the wild type, RNAi-1 and RNAi-2 transgenic lines is shown; the data are averaged (+SD), n=8; * represents the wild type and RNAi-1 or RNAi through Student's t test The difference between -2 was statistically significant (*, P<0.05; **, P<0.01).

Figure 5 shows photographs of plant heights of wild-type and overexpressed transgenic lines (35S::N- CLL1# 1 and 35S::N-CLL1#2).

Figure 6 shows that CLL1 controls genes associated with cell proliferation and cell elongation in the petiole and shaft; among them, Figure 6A shows the expression of related genes (MtCYCs and MtEXPs) revealed by RNA-seq analysis of petiole of cll1 mutants. ); Figure 6B and Figure 6C show the relative expression levels of MtCYCs in the petiole and leaf axis of wild type and cll1 mutants, respectively; Figure 6D-Figure 6I show the petiole and leaf axis of wild type and cll1 mutants, respectively The relative expression levels of MtEXPs, in Figure 6B-Figure 6I, the data are averaged (+SD), n=3, the MtGAPDH gene is used as an internal control, and the expression level values of MtCYCs and MtEXPs in the wild type are set to 1; *Represents statistically significant differences by Student's t-test (*, P<0.05; **, P<0.01), and the above experiments were repeated three times with similar results.

Detailed ways

The present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. Apparently, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art belong to the protection scope of the present invention.

experimental method

1. Plant growth conditions

All experiments were carried out in the greenhouse under the following conditions: 16 hours/8 hours day and night (temperature is 22°C/18°C), light intensity is 150 μEm-2 seconds-1, relative humidity of 35%-40%, and the plants are fully conditioned. Water and apply 0.1% water soluble fertilizer (Peters Professional Allrounder, 20-20-20+TE) every 2 weeks.

2. Scanning Electron Microscope (SEM)

The mature tissues (petioles, leaf rachis and internodes) of 7-week-old plants were collected, vacuum infiltrated 3 times in FAA fixative solution (5% formaldehyde, 5% acetic acid and 50% ethanol) for about 30 minutes, and then kept at room temperature for 72 Hour. Then in 50% ethanol (30 minutes), 60% ethanol (30 minutes), 70% ethanol (30 minutes), 80% ethanol (30 minutes), 85% ethanol (15 minutes), 90% ethanol Dehydration in ethanol (15 minutes), 95% ethanol (15 minutes), 100% ethanol (15 minutes) and 100% ethanol (1 hour). Finally, SEM was performed (He et al., 2020). All samples were tested on a Zeiss EVO LS10 (Carl Zeiss, Oberkochen) with an accelerating voltage of 5 kV. Cell length and cell number were calculated with Image J software.

3. RNA extraction and real-time fluorescent quantitative PCR (qRT-PCR)

Total RNA was extracted from various tissues using RNeasy Mini Kit (Tiangen). Use 1.5 μg of total RNA by

cDNA synthesis was performed with a 1st strand cDNA synthesis kit (Takara, Shiga, Japan). Using 25ng-50ng of RNA as a template, qRT-PCR was performed on a Roche LightCycler480II instrument (Zhang et al., 2020). The PCR assay was set up as: Stage 1 at 95°C (3 minutes); Stage 2 (28 cycles), 95°C (30 seconds), 58°C (30 seconds), 72°C (2.5 minutes); Stage 3, 72 CLL1 transcripts were amplified by RT-PCR at °C (5 minutes). MtGADPH was used as an internal control and three biological replicates were performed. The primer sequences used are shown in Table 1.

Table 1. Primer sequences

*FW is forward primer, Rv is direction primer

4. Plasmid construction

The CLL1 coding sequence from the wild-type cDNA was PCR amplified and then cloned into the Pm1I and BstEII sites of the pCAMBIA3301NM vector to generate plasmid p35S::GFP-CDS. To generate the plasmid p35S::GFP-CDS ^K440M , the N-fragment and C-fragment of CLL1 were respectively PCR amplified from the plasmid p35S::GFP-CDS (Xu et al., 2018). The N-segment and C-segment of CLL1 were recombined by overlap extension PCR, and then the recombinant fragment was cloned into the Pm1I and BstEII sites of pCAMBIA3301NM vector to generate plasmid p35S::GFP-CDS ^K440M . The natural promoter of 4071 bp from the wild type was PCR amplified and then cloned into the BamH1 and NcoI sites of the pCAMBIA3301 vector to generate plasmid pCLL1::GUS. The same sequence from plasmid pCLL1::GUS was PCR amplified and cloned into the HindIII and BamHI sites of the pGreenII 0800-Luc vector, generating plasmid pCLL1::LUC. To generate plasmid pCLL1-intron::LUC, the native promoter from plasmid pCLL1::GUS was PCR amplified, and the first intron of CLL1 from wild-type gDNA was PCR amplified. The self-promoter and the first intron of CLL1 were amplified by PCR in a fusion reaction, and then the recombinant fragments were cloned into pCAMBIA3301NM and pGreenII 0800-Luc vectors, respectively. To generate plasmid pYLRNAi.2-CLL1, a non-conserved region (321 bp) from the C-terminus of CLL1 from wild-type cDNA was PCR amplified and cloned into pYLRNAi.2 vector. The method is referred to the prior art (Hu and Liu, 2006).

Except for pYLRNAi.2-CLL1, all constructs used in this study were prepared using a one-step cloning kit (Vazyme). The recombinant plasmid was introduced into Agrobacterium tumefaciens EHA105. The primer sequences used are shown in Table 1.

5. Transactivation Activity Analysis

The CLL1-p, CLL1-p-intron 1, AtTRP1-p, and AtTRP1-p-intron 1 constructs were infiltrated into 3-week-old tobacco leaves by Agrobacterium tumefaciens EHA105. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were recorded 2 days after infiltration using a dual-luciferase assay kit (Promega) (He et al., 2020).

6. RNA sequencing

Petioles at stage 7 (primordia) were collected from wild-type and cll1 mutant plants. Total RNA was extracted using Trizol kit (Invitrogen, Carlsbad, CA, USA), and RNA quality was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Ca, USA) and agarose gel electrophoresis. Use short cDNA fragments (200bp-700bp) to assemble and prepare RNA sequencing libraries, and perform sequencing on Illumina Hiseq2500 (Guangzhou Jidio Biotechnology Co., Ltd.). After removing low-quality bases and adapters, high-quality clean reads were mapped to Mt4.0 ( http://www.medicagogenome.org/ ) of the Medicago truncatula genome using HISAT2.2.4 software (Kim et al., 2015) . Differentially expressed genes were analyzed and separated by DESeq2 software (Love et al., 2014) and Edger software (Robinson et al., 2010), and the results showed a fold change ≥ 2 and a false discovery rate < 0.05.

Experimental Results and Analysis

1. Isolation of cll1 mutants

The inventor used forward genetic screening to screen a mutant with shortened petiole and leaf axis from the fast neutron mutagenesis mutant library of Medicago truncatula (A17) at the Noble Research Institute in the United States strain (FN8157), the mutant also exhibited a semi-dwarf phenotype. The genetic test verified that the mutant is a mutant controlled by a recessive homozygous mononuclear gene, so the inventor named the mutant clustered leaf 1 (cll1). Except for the comparison with the RNAi transgenic line (R108), the wild type (WT) described herein is A17 ecotype.

2.Characterization of cll1 mutants

The inventors found through experimental research that, compared with the wild type, the petiole and rachis lengths of the cll1 mutant are different, and the cell elongation and proliferation of the petiole and rachis are abnormal.

In general, cll1 mutants have the following characteristics:

①Shortened petiole and rachis cell length: Compared with the wild-type Medicago truncatula, the cell length of the cll1 mutant was significantly shortened through cytological observation.

②The number of petiole and leaf axis cells decreased: the calculation results showed that the number of petiole and leaf axis epidermal cells in the cll1 mutant was also significantly lower than that of the wild type.

③Shortening of plant height: The inventors also observed shortening of cll1 internode length, which may be caused by abnormal cell proliferation through cytological observation.

In summary, it can be seen that the change in the length of the lateral organs of the cll1 mutant is mainly caused by the abnormal elongation or abnormal proliferation of the cells in the lateral organs.

3. CLL1 gene cloning and gene expression

①Material collection and backcrossing: The cll1 mutant was crossed with the wild type (A17), and F ₁ was the wild type phenotype. A large number of seeds (F ₂ ) of self-pollinated F ₁ plants were collected for further isolation. The ratio of mutant to wild type in F ₂ plants was 34:100 (1:3), indicating that CLL1 is a single recessive nuclear gene. ② Map-based cloning: Using map-based cloning technology, 699 and 1632 homozygous mutants segregated by F ₂ (Fig. 1A) were used to gradually map the CLL1 gene to the simple repeat sequence (SSR) marker fgp2468 and fgp2468 on chromosome 1. There is a 120Kb interval between fgp2472, and there are 17 candidate genes in this interval.

③ Determination of candidate genes: the inventors identified 17 candidate genes in the 120Kb candidate interval. Transcriptome sequencing was further performed on wild-type and mutant petioles at the seventh developmental stage, and it was found that only 3 genes expressed significantly changed in this interval, and only the 13th gene (Medtr1g100250) was significantly down-regulated. Therefore, we Medtr1g100250 in the cll1 mutant was taken as the only candidate gene. First, the inventors found through sequencing that the first intron of the gene was completely deleted (Fig. 1C-Fig. 1D), and there was no difference in other genome sequences except the first intron. Consistent with the RNA-seq data, transcripts of the complete coding sequence (CDS) of Medtr1g100250 were barely detectable by RT-PCR analysis (Fig. 1E), suggesting disrupted expression of Medtr1g100250 (Fig. 1E). qRT-PCR analysis showed that the transcript expression level of Medtr1g100250 in the wild type was about 12 times higher than that in the mutant, and the expression level of Medtr1g100250 in the mutant was significantly lower than that in the wild type (Fig. 1F). By comparing the coding sequences of wild-type and cll1 mutants, it was found that the coding sequence of Medtr1g100250 has not changed.

To determine that the first intron of Medtr1g100250 promotes gene expression. The inventors also tested using a recombinant LUC reporter construct in which LUC was fused downstream of the 4 kb CLL1 promoter and the 471 bp Arabidopsis TRP1 promoter and included the first intron ( FIG. 2A ). In both sets of experimental results, the first intron significantly stimulated the activity of the LUC reporter gene (Fig. 2C and Fig. 2E).

Therefore, the first intron is necessary for the normal expression of Medtr1g100250, and cll1 is a mutant whose expression level may decrease due to the deletion of the first intron of Medtr1g100250.

④ Genetic verification: In order to prove that the abnormal expression of Medtr1g100250 is the cause of the cll1 mutant phenotype, the inventors tried to find a mutant with an insertion of Medtr1g100250 from the Tnt1 mutant library of Medicago truncatula through reverse screening analysis, but they did not find it. Then, in order to further confirm that the defect of cll1 phenotype is related to the expression of Medtr1g100250 gene, the inventors suppressed the expression of Medtr1g100250 by RNAi (RNA interference technology). p35S::CLL1-RNAi-1 (abbreviated as RNAi-1) and p35S::CLL1-RNAi-2 (abbreviated as RNAi-2) transgenic lines had short petioles and rachis similar to cll1 plants (Fig. 4A) , and the expression level of Medtr1g100250 was significantly reduced (Fig. 3D). SEM and transgenic statistical results showed that RNAi-1 and RNAi-2 plants had abnormal cell elongation and cell proliferation in the petiole and rachis, similar to wild-type and cll1 plants (Fig. 3B-Fig. 3C and Fig. 3F-Fig. 3K) . In addition, except for the petiole and rachis, the plant height of the transgenic lines was also significantly different from that of the control group (Fig. 4). On the other hand, the inventors deleted the N-terminal inhibitory domain of the CLL1 gene, and named the coding sequence after deleting the N-terminal inhibitory domain as N-CLL1. Then connect this segment of sequence to 35S promoter to drive this segment of sequence, so that Medtr1g100250 gene is overexpressed. The petioles and rachis of the overexpressed transgenic plants (35S::N- CLL1# 1 and 35S::N-CLL1#2) were significantly elongated relative to the wild type (Fig. 5).

These results confirmed that Medtr1g100250 is the CLL1 gene, and the cll1 mutant is caused by the down-regulation of CLL1 gene expression. Mutations in the CLL1 gene lead to shortening of the lateral organs, whereas overexpression of the CLL1 gene leads to elongation of the lateral organs.

4. CLL1 expression pattern and protein subcellular localization

Evolutionary analysis and amino acid sequence comparison analysis showed that CLL1 protein has a highly conserved kinase domain, indicating that CLL1 protein can interact with other related proteins through the kinase domain to regulate plant growth and development.

In addition, qRT-PCR results showed that CLL1 was highly expressed in tissues such as young leaves, fruit clips, stems, and pollinated seeds, and was also expressed to a certain extent in petioles and leaf rachis (Fig. 6D).

Under the control of cauliflower mosaic virus 35S promoter (CaMV35S), the fusion protein of CLL1 and green fluorescent protein (GFP-CLL1 ^K440M ) was transiently expressed in tobacco leaves to determine the subcellular localization of CLL1 protein. The detected structure showed that the GFP-CLL1 ^K440M fusion protein was located in the cytoplasm and nucleus of tobacco epidermis. These data suggest that the CLL1 protein is a conserved MAPKKK protein.

5. CLL1 participates in signaling pathways to regulate the development of petiole and leaf axis

Existing literature shows that YODA, as a key link in the phosphokinase pathway, receives upstream signals, then phosphorylates downstream MKK family protein kinases, and then transmits the signals. And in Arabidopsis and rice, the two pathways YODA-MKK4/5-MAPK3/6 and OsMKKK10-OsMKK4-OsMAPK6 have jointly proved that MKK4/5 is the key protein for downstream signaling. However, existing literature shows that only one MtMKK4 has evolved in M. truncatula to replace AtMKK4 and AtMKK5 in Arabidopsis. And it has been proved that there is a protein interaction between MtMKK4 and downstream MtMAPK3/6 in regulating the nodulation and fertility of Medicago truncatula. Therefore, the inventors speculate that the CLL1-MtMKK4 cascade may be a conserved signaling pathway in Medicago truncatula.

In order to perfect this signaling pathway in Medicago truncatula, the inventors confirmed the interaction between CLL1 protein and MtMKK4 through yeast two-hybrid (Y2H), bimolecular fluorescence complementation and fusion protein deposition techniques. In addition, the inventors also performed a protein phosphorylation test in vitro to prove that CLL1 protein can phosphorylate MtMKK4. Therefore, the CLL1-MtMKK4 signaling pathway may be involved in the positive regulation of the development of petiole and leaf rachis of M. truncatula.

6. CLL1 affects the cell cycle and regulates gene expression

The development of plant organs is regulated by two aspects of cell elongation and cell division. In order to analyze the mechanism by which CLL1 regulates the elongation of petiole and leaf axis, the inventors collected the petioles of wild-type and cll1 plants at the seventh stage of development for RNA-seq. Compared with wild-type petioles, 1218 genes were significantly up-regulated and 1187 genes were down-regulated in the cll1 mutant. In addition, the results of KEGG pathway analysis showed enrichment of genes related to brassinosterol biosynthesis (ko00905) and MAPK signaling pathway (ko04016), which are involved in plant development.

The results of RNA-seq and qRT-PCR jointly proved that the expression levels of MtCYC2; 2 and MtCYC2; 3 and other cell cycle-related genes were all significantly down-regulated in the petiole of cll1 plants (Figure 6B and Figure 6C), and MtEXP13, MtEXP11, MtEXPB3 and MtXTH8 The expression levels of genes related to cell elongation were also all down-regulated ( FIG. 6D , FIG. 6E , FIG. 6G and FIG. 6H ). Because the development process of the leaf rachis is similar to that of the petiole, it was found that the gene expression of MtCYC2;3 in the leaf rachis was significantly down-regulated (Fig. - Figure 6I).

These results suggest that CLL1 affects core cell cycle and cell elongation gene expression in both the petiole and rachis of M. truncatula, thereby regulating organ size.

7. CLL1 and its corresponding legume orthologous genes

Phylogenetic analysis showed that CLL1 has conserved homologs in other important leguminous crops, such as chickpea, pea, soybean, kidney bean, red bean, groundnut, vine, mung bean, clover, alfalfa and lotus root. Source genes (see Table 2).

Table 2. CLL1 gene in Medicago truncatula and homologous gene of CLL1 legume in related species

Evidence for the involvement of CLL1 and its corresponding legume orthologs in the regulation of lateral organ elongation in different species is as follows:

1) According to the evolutionary analysis of CLL1 protein and homologous proteins in corresponding species, it can be seen from the evolutionary tree that all proteins have homology and gather on corresponding branches, which on the one hand shows the conservation of their functions;

2) Analyze the protein conserved domains of CLL1 protein and homologous proteins in corresponding species. The analysis results show that: both CLL1 and its corresponding legume homologous proteins have a conserved kinase domain; a specific family of conserved functions According to the existing literature, the two pathways YODA-MKK4/5-MAPK3/MAPK6 and OsMKKK10-OsMKK4-OsMAPK6 play a role in regulating pedicel elongation, stomatal formation, There are conserved mechanisms in plant architecture formation, tap root, lateral root and seed elongation. Therefore, the protein structure conservation of CLL1 and its homologous proteins may also participate in similar signaling pathways in alfalfa to regulate the functional conservation of organ development;

It can be seen that CLL1 or its homologous genes have positive regulatory functions on lateral organs in multiple species, which is the most direct evidence for the functional conservation of CLL1 and its homologous genes.

By regulating the CLL1 gene and its homologous genes, the elongation of the lateral organs of the plant can be directly regulated, the ratio of leaves/petiole of the plant can be greatly increased, and the plant height of the plant can be adjusted at the same time. For legumes, increasing the leaf/petiole ratio can increase photosynthetic efficiency per unit area and thus increase yield. At the same time, for pastures, the yield of edible parts can be greatly increased (the protein content of petioles in pastures is much lower than that of leaves, and leaves are the main edible parts). The semi-dwarf plant type can be reasonably densely planted for leguminous crops, thereby increasing its yield. Therefore, CLL1 and its homologous genes have great application potential and value in molecular breeding.

Table 3. Related sequences

Claims (10)

1. A mutant Medicago truncatula CLL1 gene is characterized in that, compared with Medtr1g100250, the first intron of the mutant Medicago truncatula CLL1 gene is deleted, and the first intron sequence of the deletion is as shown in SEQ ID NO.1 Show.
2. The mutated Medicago truncatula CLL1 gene according to claim 1, wherein the nucleotide sequence of the mutated Medicago truncatula CLL1 gene is as shown in SEQ ID NO.2, and the CLL1 protein sequence encoded by it is as shown in SEQ ID NO. 3.
3. According to the application of the CLL1 protein in regulating the elongation of plant lateral organs according to claim 2, it is characterized in that the regulation is positive regulation.
4. A kind of Medicago truncatula cll1 mutant, it is characterized in that, compared with wild type, described cll1 mutant comprises the mutated Medicago truncatula CLL1 gene of claim 1, and its nucleotide sequence is as shown in SEQ ID NO.2 .
5. A method for improving plants, characterized in that the method comprises downregulating the expression of CLL1 gene, such as removing the first intron of the CLL1 gene or reducing the expression of CLL1 gene by using RNA interference technology.
6. The method according to claim 5, characterized in that, the fragment used for RNA interference is as shown in SEQ ID NO.4.
7. A fusion expression vector, the fusion expression vector includes the gene sequence to be expressed and the first intron of the CLL1 gene inserted and replaced.
8. According to the fusion expression vector of claim 7, the gene sequence to be expressed is as shown in SEQ ID NO.5; the first intron sequence of the CLL1 gene is as shown in SEQ ID NO.1.
9. The legume orthologous protein of the CLL1 protein of claim 2, wherein the amino acid sequence of the protein is as shown in SEQ ID NO.6-20, and the legume plant comprises: chickpea (Cicer arietinum ), pea (Pisum sativum), soybean (Glycine max), kidney bean (Phaseolus vulgaris), red bean (Vigna angularis), groundnut (Arachis hypogaea), vine peanut (Arachis duranensis), mung bean (Vigna radiata) clover (Trifolium pratense ), alfalfa (Medicago sativa) and japonicus (lotus japonicus).
10. The gene encoding the leguminous orthologous protein of the CLL1 protein according to claim 7, wherein the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.21-35.

Images