CN116769792B - Phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof - Google Patents
Phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof Download PDFInfo
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Abstract
The invention discloses a phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof, wherein the Mao Zhujing stem elongation related gene PheLBD12 has a nucleotide sequence shown as SEQ ID NO. 1. The invention provides a Phyllostachys pubescens PheLBD12 gene related to regulating and controlling stem elongation for the first time, and a coding protein and application thereof. The PheLBD12 gene over-expression vector is transferred into wild rice through agrobacterium-mediated rice callus, and the result shows that the over-expression strain has obviously changed strain height compared with the wild strain. The invention provides new gene resources and breeding resources for revealing the regulation and control mechanism of Mao Zhujing stalk elongation.
Description
Technical Field
The invention relates to the technical field of plant molecular biology, in particular to a phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof.
Background
Bamboo is of interest to researchers as a worldwide important non-wood product in its unique rapid growth pattern. Previous researches on the high growth of bamboo plants mainly focus on the aspects of high growth rhythm of bamboo, cellular structure, physiological and biochemical changes, hormone regulation and the like of bamboo shoots. The high growth of bamboo generally follows a "slow-fast-slow-stop" growth rhythm. Tissue anatomic observations indicate that the rapid growth of bamboo stalks is the result of the co-action of the apical meristem and the intervening meristem of each node, cell division being the basis of internode elongation, cell elongation being the primary cause of internode elongation. Proteins and carbohydrates are important nutrients and energy required for differentiation division, elongation and maturation of cells during high growth of bamboo. The plant hormone plays an important role in the plant growth process, and the synergistic regulation and control effect of the plant hormone is important for the rapid growth of the bamboo shoots. Bamboo growth rate is positively correlated with gibberellin and auxin content, cell division rate is significantly positively correlated with cytokinin concentration during internode elongation, and is negatively correlated with abscisic acid concentration.
With the intensive and histological development of high growth research on bamboo, many functional genes related to high growth of bamboo are discovered, and the functional genes are involved in synthesis and degradation of phytohormone and the like by regulating cell division and differentiation, elongation and maturation, so that the high growth process of bamboo is affected. C in green bamboo 2 C 2 The zinc finger protein BohLOL1 is differentially expressed in various bamboo shoot tissues, and the gene can be up-regulated in the bamboo shoots in the process of growing with high bamboo stalks, which indicates that BohLOL1 is possibly related to high bamboo growth. The SINAT protein PpSINA in the Lei bamboo is an Arabidopsis SINAT5 homologous gene. PpSINA is expressed in all organs of bamboo shoots and is significantly higher in whip buds than in shoots and shoots, whip buds being the major sites of auxin synthesis in shoots. It was therefore inferred that PpSINA might regulate the growth and development of bamboo shoots by participating in auxin signaling. PheIAA1, pheIAA2 and PheIAA3 of AUX/IAA family in Phyllostachys pubescens are expressed at each tissue site of the bamboo shoot, and the expression level of PheIAA1 is in an ascending trend in the whole growth process of the bamboo shoot; the expression level of PheIAA2 reaches the maximum value at the end of the development of the bamboo shoots; the expression level of PheIAA3 is reduced along with the growth and development of the bamboo shoots, which shows that the three genes possibly participate in the rapid growth regulation of the bamboo shoots. In addition, SAUR (small auxin up RNA) protein PheSAUR in phyllostachys pubescens is expressed in all parts of the phyllostachys pubescens, the expression level of the basal part is not changed greatly, the expression level of the middle part and the top part is high, and especially the highest expression level is in the middle part of the phyllostachys pubescens at the height of 3-12m, which indicates that PheSAUR possibly participates in the rapid growth process of phyllostachys pubescens and plays an important role in the internode elongation. However, these results are more of the related studies of gene expression regulation, and there are still few more studies on the molecular mechanism of functional gene regulation in Mao Zhugao growth.
Disclosure of Invention
The invention aims to provide a Mao Zhujing stalk elongation related gene PheLBD12 capable of changing the plant height of rice and application thereof.
In order to solve the problems in the prior art, the invention provides the following technical scheme: the gene PheLBD12 related to the elongation of the moso bamboo stalks has a nucleotide sequence shown as SEQ ID NO.1, and the gene PheLBD12 related to the elongation of the Mao Zhujing stalks has a nucleotide sequence shown as SEQ ID NO. 1.
The protein encoded by the Mao Zhujing stalk elongation related gene PheLBD12 is the protein as shown in the following (1) or (2):
(1) A protein consisting of the amino acid sequence of SEQ ID No.2 in a sequence table;
(2) And (3) a protein which is obtained by substituting and/or adding one to ten amino acid residues in the amino acid residue sequence of SEQ ID No.2 in the sequence table and has PheLBD12 function of a phyllostachys pubescens stem elongation related gene.
The invention relates to a plant over-expression vector or recombinant bacterium containing the Mao Zhujing stalk elongation related gene PheLBD 12.
Further, the plant over-expression vector is a plant expression vector of pCAMBIA1301a-PheLBD12 with a 35S promoter, the PheLBD12 gene and a terminator connected in sequence in a multiple cloning site region.
The invention also provides a host bacterium containing the plant over-expression vector.
The invention relates to application of a Mao Zhujing stalk elongation related gene PheLBD12 in regulating stalk elongation.
The primer pair for cloning the Mao Zhujing stalk elongation related gene PheLBD12 comprises an upstream primer and a downstream primer, wherein the upstream primer has a nucleotide sequence shown as SEQ ID No.3, and the downstream primer has a nucleotide sequence shown as SEQ ID No. 4.
The genetically engineered host cell of the invention contains the plant over-expression vector or the Mao Zhujing stalk elongation related gene PheLBD12 sequence integrated with an exogenous source in the genome.
The beneficial effects are that: the invention provides a Phyllostachys pubescens PheLBD12 gene related to regulating and controlling stem elongation for the first time, and a coding protein and application thereof. The PheLBD12 gene over-expression vector is transferred into wild rice by an agrobacterium-mediated rice callus method, and the result shows that the over-expression strain has obvious change in plant height compared with the wild strain. The result provides a theoretical basis for researching Mao Zhujing stalk elongation.
Drawings
FIG. 1 is a domain division diagram of a protein encoded by the PheLBD12 gene of the present invention.
FIG. 2 is a bar graph showing the results of analysis of different tissue expression patterns of the PheLBD12 gene of the present invention.
FIG. 3 is a screening chart of positive seedlings of the transgenic rice over-expressing PheLBD12 of the present invention, and FIG. 3A is a PCR detection of the target gene of the present invention. Negative control; positive control: OE1 to OE7: different transgenic lines; FIG. 3B shows GUS staining of leaves of different strains according to the invention.
FIG. 4 is a phenotypic analysis of transgenic rice over-expressing PheLBD12 according to the present invention; FIGS. 4A-4B are graphs showing plant height phenotypes of wild type rice and PheLBD 12-overexpressing transgenic rice in the growing period of the present invention; FIGS. 4C-4D are graphs showing statistical analysis of internode length of wild-type rice and PheLBD 12-overexpressing transgenic rice according to the present invention.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Example 1
The gene PheLBD12 related to the elongation of the moso bamboo stalks has a nucleotide sequence shown as SEQ ID NO.1, and the gene PheLBD12 related to the elongation of the Mao Zhujing stalks has a nucleotide sequence shown as SEQ ID NO. 1.
The protein encoded by the phyllostachys pubescens leaf shape-related gene PheLBD12 is the protein as shown in the following (1) or (2):
(1) A protein consisting of the amino acid sequence of SEQ ID No.2 in a sequence table;
(2) And (3) a protein which is obtained by substituting and/or adding one to ten amino acid residues in the amino acid residue sequence of SEQ ID No.2 in the sequence table and has PheLBD12 function of a phyllostachys pubescens stem elongation related gene.
The invention relates to application of a phyllostachys pubescens leaf shape related gene PheLBD12 in regulating rice plant height.
The invention relates to a plant over-expression vector or recombinant bacterium containing the Mao Zhujing stalk elongation related gene PheLBD 12.
The plant over-expression vector is a pCAMBIA1301a-PheLBD12 plant expression vector with a 35S promoter, the PheLBD12 gene and a terminator connected in sequence in a multiple cloning site region.
The invention also provides a host bacterium containing the plant over-expression vector.
The primer pair for cloning the Mao Zhujing stalk elongation related gene PheLBD12 comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.
The invention relates to a genetically engineered host cell, which contains the plant over-expression vector or the gene PheLBD12 sequence of the Mao Zhujing stem elongation related gene integrated with an exogenous source in the genome.
Test example 1
1 Material
The methods used in this example are conventional methods known to those skilled in the art unless otherwise indicated, and the materials such as reagents used are commercially available products unless otherwise indicated.
2 method
2.1 protein sequence analysis of PheLBD29 Gene
The PheLBD12 gene was searched using the Moso bamboo database and the corresponding protein sequence (SEQ ID No. 2) was found, and the PheLBD12 protein sequence domain was analyzed by NCBI-CDD website, the PheLBD12 protein sequence comprising the LOB domain. The PheLBD12 protein was shown to belong to the LOB family (FIG. 1).
2.2 analysis of the expression patterns of different tissues of the PheLBD29 Gene
Taking the Top (Top portion), the Middle (Middle portion) and the bottom (Lower portion) of bamboo shoots (0.2, 1.5, 3 and 6.7 m) with different heights of moso bamboo, quickly freezing in liquid nitrogen after each sample is taken, and then freezing in a refrigerator at-70 ℃ for extraction and use in later-period RNA.
RNA extraction procedure was carried out with reference to the TaKaRa MiniBEST Universal RNA Extraction Kit kit (TaKaRa: 9767). The reverse transcription step refers to PrimeScript TM RT reagent Kit (Perfect Real Time) Kit (TaKaRa: RR 037Q). Fluorescent quantitative PCR primers: pheLBD12F 5' -CGGATCGTGAACCCCATCTA-3; pheLBD12F 5' -GTCCTTGTCGAGCTTGGAGA-3.
Reference gene: TIP41F:5' -AAAATCATTGTAGGCCATTGTCG-3; TIP41R:5'-ACTAAATTAAGCCAGCGGGAGTG-3'.
The quantitative reaction uses TaKaRa quantitative kit, the reaction system is 20 mu L, each component is 7.0 mu L of dye mixed solution, the cDNA template is 1.5 mu L, each of the upstream primer and the downstream primer (10 mu mol/L) is 0.5 mu L, and finally deionized water is added to 20 mu L. The PCR parameters were as follows: 95 ℃ for 10min;95 ℃ for 15s;60 ℃ for 1min, and 40 cycles. After the reaction is finished, the product is heated, and a dissolution curve of the product is obtained. By using 2–ΔΔ CT[ Δ Ct=ct target gene-CT reference gene. ΔΔ CT＝ Δ post-CT treatment Δ CT control]The method performs data processing.
Three biological replicates were measured per experiment, at least three experimental runs were repeated, and the measurement results are shown in FIG. 2, in which it can be seen that PheLBD12 gene was expressed in bamboo shoots of different heights, and the expression level was highest at the bottoms of the bamboo shoots of different heights.
2.3 cloning of the PheLBD12 protein coding sequence of Phyllostachys Pubescens
And (3) taking bamboo shoot cDNA with the height of 0.2m of moso bamboo as a template, designing a primer according to the published gene sequence of the moso bamboo genome database and combining with a multiple cloning site of a cloning vector, and performing PCR amplification to obtain a PCR amplification product.
The primer sequences were as follows:
PheLBD12F:5’-GGGGTACCCCATGAGGCTGAGCTGCAAC-3’；
PheLBD12R:5’-CGGGATCCCGCTACAGGAACCGGAGGCC-3’
the PCR reaction system is shown in Table 1:
TABLE 1
The PCR reaction conditions were: pre-denaturation: 98 ℃ for 5min; denaturation: 98 ℃ for 10s; annealing: 55 ℃ for 5s; extension: 30s at 72℃for 33 cycles; total extension: and at 72℃for 10min.
After the reaction is finished, absorbing the PCR product to carry out 2% agarose gel electrophoresis, detecting and cutting gel in a gel imaging system, recovering by using an agarose gel DNA recovery kit, and sending the recovered product to Shanghai industrial company for sequencing, wherein the sequencing result is as follows: the DNA sequence of SEQ ID NO.2 in the sequence table shows that the DNA sequence consists of 921bp bases, and the DNA sequence is completely consistent with the published coding sequence of the PheLBD12 protein of Phyllostachys pubescens.
2.4 construction of the overexpression vector pCAMBI1301a-PheLBD12
Using two restriction endonucleases KpnI and BamHI to carry out double enzyme digestion on pEASY Blunt simple Cloning Vector recombinant plasmid which contains PheLBD12 gene coding sequence and over-expression vector pCAMBIA1301a plasmid which are correctly sequenced in a water bath kettle at 37 ℃ for 3 hours; the table of the double cleavage system is shown in Table 2:
TABLE 2
After double digestion, the DNA was detected by 2% agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit. The obtained 1301a vector large fragment and the target gene small fragment were ligated by T4DNA ligase, and reacted at 25℃for 20min, and the T4 ligase reaction system is shown in Table 3:
TABLE 3 Table 3
Lightly pumping the connection product into 50 mu L of EHA105 agrobacterium competent cells, ice-bathing for 7min, quick-freezing with liquid nitrogen for 3min, heat-shocking with 37 ℃ metal bath for 3min, adding 500 mu L of YEP liquid culture medium, and culturing at 28 ℃ for 2-3 h; centrifuging for 30s, discarding the supernatant, adding 100. Mu.L of YEP liquid medium, re-suspending the cells, plating on a YEP solid plate containing 50. Mu.g/mL Kan and 30. Mu.g/mL Rif, and culturing at 28 ℃ for about 48 hours; single colonies growing on the plates were picked, inoculated into YEP liquid medium containing 50. Mu.g/mL Kan and 30. Mu.g/mL Rif, shaken for 48h, plasmids were extracted, and verified by PCR and double restriction enzyme digestion, respectively.
2.5 obtaining and screening of transgenic Rice overexpressing PheLBD12
1. Obtaining of transgenic rice overexpressing PheLBD 12:
sterilizing mature and full rice seeds by sodium hypochlorite, spreading on an induction culture medium, and growing light yellow callus for about two weeks; placing the seeds with the callus into an agrobacterium suspension, slightly shaking for 30 minutes, pouring out bacterial liquid, spreading the callus on sterile filter paper, and sucking the bacterial liquid around the callus; then placing the mixture into an infection culture medium, and carrying out dark culture at 22 ℃ for 36 hours; selecting infected callus, and cleaning with pure water until bacterial liquid on the surface is washed away; placing the cleaned callus on a screening culture medium for culturing, and growing new yellowish callus with resistance after 30 days; selecting new pale yellow callus with resistance on a differentiation medium, and culturing for about 30 days to grow resistant callus seedlings; and then transferring to rooting culture medium for continuous culture until the young seedling grows to the bottle mouth, opening sealing film, pouring sterilized pure water, starting to train the seedling, and transplanting to field after about 3 days.
2. Screening positive seedlings:
the transgenic rice DNA of the PheLBD12 gene was first extracted, and the genome extraction was carried out in accordance with the Rapid Plant Genomic DNA Isolation Kit kit (Sangon Biotech: B518231-0050). The above-mentioned extracted genomic DNA was used as a template, and primers PheLBD12-F and PheLBD12-R were used for PCR molecular detection, and the PCR reaction was 2.3. After the reaction, the PCR product was taken and subjected to 2% agarose gel electrophoresis, and then observed in a gel imaging system (FIG. 3A).
After PCR detection, the positive young leaves are further sheared to carry out Gus histochemical staining. The cut leaves were placed in a 1.5mL Dof tube, and Gus was added to dye the leaves, and the dye solution was used to submerge the leaves completely at 37℃for 12 hours. Then, alcohol decolorization was performed to observe the staining of the leaf, as shown in FIG. 3B.
2.6 plant height phenotype analysis of PheLBD12 over-expressed transgenic Rice
Simultaneously, wild type and transgenic rice are planted in a greenhouse, and after four weeks, phenotype differences are observed through photographing. As a result, as shown in FIG. 4, it can be seen that the over-expressed strain is significantly shorter in height than the wild-type strain (FIGS. 4A-B). And meanwhile, carrying out statistical phenotype analysis on the internode length of the wild rice and the PheLBD12 transgenic rice. As a result, it was found that the internodes of the transgenic rice were significantly shorter than those of the wild-type rice, resulting in the transgenic rice having a significantly shorter plant height than that of the wild-type rice (FIGS. 4C-D).
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.
Claims (1)
1. The application of the phyllostachys pubescens stem elongation related gene PheLBD12 in the regulation of rice plant height is characterized in that: the plant height of the transgenic rice which is over-expressed with PheLBD12 is shortened, and the nucleotide sequence of the gene PheLBD12 related to the elongation of Mao Zhujing stalk is shown as SEQ ID NO. 1.
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