CN110938617B - Lilium regale LrPAL-1 gene and application thereof - Google Patents

Abstract

The invention discloses Lilium regaleLrPAL‑1Genes and uses thereofLrPAL‑1The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The invention will compriseLrPAL‑1The overexpression vector of the gene is transferred into tobacco, the lignification degree of the obtained transgenic tobacco plant is increased, the lodging resistance of the plant is improved, and the plant can be particularly used for maintaining the upright state of fresh cut flowers of lily; the transgenic plants have obvious inhibition effect on pathogenic bacteria of botrytis cinerea and alternaria alternata; meanwhile, the invention proves lilium regale through functional genomics researchLrPAL‑1The gene has important biological function of changing the shape of plant leaf. The invention provides an important target for the molecular genetic improvement of lily plants, has important value and significance for the cultivation and production of new plant varieties with ornamental value and landscape ecological value, and also provides technical support for cultivating new lily germplasm with double values of lodging resistance and pathogenic bacterium resistance.

Description

Lilium regale LrPAL-1 gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to Lilium regaleLrPAL-1Genes and their use.

Background

Lily is a perennial herb-root plant distributed all over the world. Lily is one of important ornamental flowers in the world and has important economic value. China is the distribution center of lilies in the world, has abundant wild lily resources, and about 55 varieties (Flora of China 24: 135-149.2000.). Lilium regale (Lilium regale)Lilium regale Wilson) is an important wild germplasm resource in Lilium of Liliaceae, and is widely distributed in Sichuan of ChinaMinjiang river basin (Yangliping and heroic character, lily resource utilization research in China [ M)]And Harbin: northeast forestry university press, 2018.11). Compared with the model plant, the growing and developing regulation mechanism of the Lilium regale has complexity and specificity. The previous research shows that Lilium regale has stronger resistance to lily mycosis and virosis, and a plurality of adversity stress related genes such as Lilium regale are obtained from Lilium regaleLr14-3-3、LrPR10、LrbZIP1AndLrWRKY1etc. (Li h.,et al., Sci. Hort. 2014, 168:9-16; He H., et al., Genes Genom. 2014, 36:497-507; Zhang N.N., Genes Genom. et al., 2014, 36:789-798; Han Q., et al.sci. Hort. 2016, 198; patent number 201610001896.4), the resistance genes are far from enough in order to enrich the gene bank, and it is worth further exploring more new high-efficiency antibacterial genes in Lilium regale.

The leaves are the main places for plants to carry out photosynthesis and are key organs for plant growth and development. The change of the leaf shape can adjust the light transmittance of the leaf, thereby changing the photosynthetic efficiency of the plant, and in addition, the leaf shape which shows a series of novel and peculiar leaf shapes on the shapes of the flower plant leaf shape and the like also has good ornamental value. In recent years, many related researches on molecular regulation mechanisms of plant leaf type growth and development are carried out, and reports show that miRNA, transcription factors, plant hormones and the like play a very important regulation role in the growth and development of leaves. Meanwhile, if the height control of the ornamental plants is not good, the ornamental plants are easy to fall down, mainly because the stems are not thick, strong, straight and weak in uprightness, and the ornamental plants are not convenient to manage, harvest and transport when used as the fresh cut flower varieties and are inconvenient to bottle insert. Therefore, the method promotes the growth and development of leaves and the lodging resistance through genetic improvement, and has important value and significance for the cultivation and production of new plant varieties with ornamental value and landscape ecological value.

Phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5) is an enzyme which exists only in plant and microbial cells and is the first step of catalyzing the metabolism of phenylalanines by linking the primary metabolism of organisms with the metabolism of phenylalaninesThe enzyme of the reaction is a key enzyme and a rate-limiting enzyme of the metabolism of the phenylpropanoids. The phenylpropanoid metabolic pathway is an important pathway in the metabolism of organisms, particularly plants, and all substances containing a phenylpropane skeleton are directly or indirectly generated by the pathway. The phenylpropanoids metabolism can generate a plurality of secondary metabolites such as flavonoid, lignin and the like, and the secondary metabolites play important roles in the growth and development, disease resistance and stress resistance of plants. Studies have shown that PAL family members vary in number among plants, e.g., 4 in ArabidopsisPALGene (Cochrane FC, davin LB, lewis NG 2014 TheArabidopsisphenylalkane ammonia lyase family: (ii) kinetic characterization of the four PAL aspects 65 (11): 1557-1564) and 5 poplar treesPALA gene; wherein the poplarPtPAL1AndPtPAL3mainly participates in the synthesis regulation and control of flavonoid, tannin and other substances; whilePtPAL2、 PtPAL4、PtPAL5High expression mainly in xylem, and may be involved in the regulation of the lignin synthesis pathway (Shi R, shuford CM, wang JP,et al., 2013. Regulation of phenylalanine ammonia-lyase (PAL) gene family in wood forming tissue of Populus trichocarpa. Planta 238(3):487-497）。

at present, lily is concernedPALThe research on the gene function is only reported, and the research finds that the musk lilyLlPAL1The gene is related to the metabolism of phenols at the base of the cut flower stem (Cao Spirodela, li hong Mei, wu Shi Cheng, etc., musk lily)LlPAL1Cloning of genes and expression analysis of genes at the base end of cut flower stems, abstract set of academic annual meeting paper 2014 of Chinese horticulture academy), and for Lilium regalePALThe gene function research is not reported. Further, in the genus Lilium, which arePALThe genes are mainly involved in the flavonoid synthesis pathway, which lilies arePALThe involvement of genes in the lignin synthesis pathway and the like is still unclear. In addition, no report is found on the study of simultaneous participation of lily PAL in plant disease resistance, lodging resistance and leaf type development.

Disclosure of Invention

Aiming at the blank existing in the prior art, the invention aims to provide Lilium regaleLrPAL-1Gene of BaiThe genetic improvement of symphytic plants provides a new lignin synthesis regulation gene, provides more selection possibilities for plant disease-resistant genetic engineering, and simultaneously provides support for improving the lodging resistance of lily plants or fresh cut flowers and the special shape of leaves by molecular means.

In order to achieve the purpose, the invention adopts the following technical scheme: lilium regaleLrPAL-1The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The invention also provides the Lilium regaleLrPAL-1The amino acid sequence of the gene-coded protein is shown in SEQ ID NO. 2.

The invention also provides a pharmaceutical composition containing the aboveLrPAL-1Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria of the genes.

The recombinant vector can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA1302, pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-inducible promoter rd29A, and the like, can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters.

The invention also provides the aboveLrPAL-1The application of the gene or the LrPAL-1 protein or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in (C1) or (C2) or (C3) as follows:

(C1) Regulating and controlling the pathogen resistance of the plant;

(C2) Regulating and controlling the development of plant leaves;

(C3) Regulating and controlling the lodging resistance of the plant.

Further, the plant pathogenic fungi are botrytis cinerea and/or alternaria alternata.

Furthermore, the plant leaf development is regulated and controlled to change the leaf form, and the proliferation and folding of the plant leaves are promoted.

The invention also provides a method for improving the disease resistance of plants or improving the lodging resistance of plants or changing the leaf morphology of plants, which comprises the step of improving the expression level and/or the activity of the LrPAL-1 protein in recipient plants.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: introducing said plant into a recipient plantLrPAL-1Gene, obtaining transgenic plants; the transgenic plant has enhanced disease resistance or enhanced lodging resistance or altered leaf morphology as compared to the recipient plant.

Further, the plant is a dicotyledonous plant or a monocotyledonous plant, preferably a lilium plant or tobacco, and the like.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention firstly clones Lilium regaleLrPAL-1The gene full-length sequence is obtained, the coded protein sequence is obtained, and the protein is expressed in tobacco, and test results show that the protein can increase the lignification degree of transgenic plants, improve the lodging resistance of the plants, and particularly maintain the upright state of fresh cut flowers of lily. Meanwhile, the protein can change the shape of the leaf and show the phenomenon of hyperplastic folds. The invention provides an important target for the molecular genetic improvement of the lily plants, and has important value and significance for the cultivation and production of new plant varieties with ornamental value and landscape ecological value.

2. The invention is proved by the analysis of antibacterial testLrPAL-1The gene has the function of improving the ability of lily plants to resist pathogenic fungi, in particular to botrytis cinerea (A), (B)Botrytis cinerea) And Alternaria alternata(Alternaria alternate) Has strong inhibiting effect and provides an effective disease-resistant gene for lily and other plant disease-resistant genetic engineering. By the method of the present invention, for the futureLays a foundation for culturing new lily germplasm with double effects of lodging resistance and pathogenic bacteria resistance. Therefore, the invention has strong application prospect and popularization value.

Drawings

FIG. 1 is Lilium regaleLrPAL-1PCR amplification electrophoretogram of gene;

FIG. 2 is an amino acid sequence alignment diagram of Lilium regale LrPAL-1 protein and a published RNA sequencing splice sequence (KX 842518.1);

FIG. 3 is a PCR detection result electrophoresis diagram of transgenic tobacco genome DNA, lanes 1-16 are T0 generation transgenic lines, M is DL2000, WT is negative control, CK is blank control, and P is positive control;

FIG. 4 is the identification and analysis of positive plants of transgenic tobacco, WT is wild type tobacco,OX-L1、OX-L7、OX-L93 represent transgenic tobacco positive lines; wherein A is shown in positive transgenic tobacco of T1 generationLrPAL-1Quantitative expression analysis of gene transcript levels; b is a PAL enzyme activity detection result diagram of the positive transgenic tobacco leaf of the T1 generation, the data analysis adopts a Duncan multiple comparison method (Duncan test), and different letters represent obvious differences (P)<0.05）；

FIG. 5 is a phenotype map of T2 generation positive transgenic tobacco and wild type tobacco; panel A is wild type tobacco plant (WT), and panel B is transgenic tobacco plant (WT)PAL-1-OX）；

FIG. 6 is a leaf phenotype plot of transgenic tobacco and wild-type tobacco; panel A shows Wild Type (WT) and transgenePAL- 1-OX) Tobacco section 3 leaf, panel B Wild Type (WT) and transgene (B)PAL-1-OX) Tobacco section 4 lamina;

FIG. 7 is a cross-sectional lignin staining analysis of transgenic tobacco and wild-type tobacco stems; WT is phloroglucinol staining pattern of cross section of 3 rd node stem of wild type tobacco,OX-L1，OX-L7andOX-L9phloroglucinol staining pattern of cross section of 3 rd node stem of 3 independent transgenic lines; the scale in the figure represents the actual length 200 μm;

FIG. 8 is the result analysis of transgenic tobacco in vitro leaf against Botrytis cinerea; wherein, the graph A is a schematic diagram of the resistance of wild tobacco and transgenic tobacco in-vitro leaves to botrytis cinerea, the graph B is a comparison of infected areas of the botrytis cinerea infected tobacco in-vitro leaves for 3 days, a Duncan multiple comparison method (Duncan test) is adopted for data analysis, and different letters show obvious difference (P < 0.05);

FIG. 9 is the analysis of the effect of transgenic tobacco in vitro leaf against Alternaria alternata; wherein, the graph A is a schematic view of resisting alternaria alternate of wild tobacco and transgenic tobacco in-vitro leaves, the graph B is a comparison of infected areas of the alternaria alternate infected with the tobacco in-vitro leaves for 7 days, a Duncan multiple comparison method (Duncan test) is adopted for data analysis, and different letters show obvious difference (P < 0.05);

FIG. 10 is an analysis of total flavone content of wild type and transgenic tobacco leaves; wherein the WT is a wild-type tobacco,OX- L1，OX-L7andOX-L9the data analysis is performed by a Duncan multiple comparison method (Duncan test) for 3 independent transgenic tobacco lines, and different letters indicate that the difference is obvious (P)<0.05）。

Detailed Description

The invention will be described in more detail with reference to specific embodiments and the drawings, but the scope of the invention is not limited thereto. In the examples, the starting materials are all common commercial products unless otherwise specified. The experimental procedures described in the examples are not specifically described, i.e., they are carried out according to conventional molecular biological experimental procedures.

Example 1 Lilium regaleLrPAL-1full-Length Gene cloning and sequence analysis

Contains Botrytis cinerea (A)Botrytis cinerea) The spore solution of the strain is used for infecting Lilium regale (bud stage), leaves at different time (6 h, 12 h, 24h and 48 h) are taken as an experimental group, and the leaves without the spore solution are taken as a control group. Adopting TRIzol ™ Plus RNA Purification Kit (12183555, invitrogen;) to extract total RNA according to the instruction steps, utilizing DNase I (18047019, invitrogen) to remove residual trace DNA, using a differential spectrophotometer to determine the concentration of RNA, and sending a part of high-quality RNA to a Huada gene BIOSEQ500 sequencing platform for sequencing analysis; the other part of RNA is stored for later use.

Through transcriptome sequencing analysis, 3 encoding Lilium regale phenylalanine ammonia-lyase (b) are preliminarily obtainedLrPAL) The gene's Unigene (Unigene 3248_ All, unigene14345_ All, unigene556_ All). Wherein the expression level of the Unigene3248_ All after responding to the induction of the botrytis cinerea is the highest and is 4.3 times of that of a control group, and the expression level is named LrPAL-1。

About 2.0. Mu.g of total RNA from Lilium regale leaf was used to synthesize first strand cDNA according to PrimeScript II first-strand cDNA synthesis kit (6210A, takara) instructions.

PCR amplification System: 2xfast pfu master Max 10ul, 1. Mu.L of forward primer (LrPAL-1-F, 10. Mu.M), 1. Mu.L of reverse primer (LrPAL-1-R, 10. Mu.M), 1. Mu.L of template (cDNA), sterile ddH ₂ Make up to 20. Mu.L of O.

The forward and reverse primers were:

LrPAL-1-F：5’-ATGGCATCCAAGGACAGCT -3’

LrPAL-1-R：5’-TCAGAGATTCCAGTACCCCCT -3’

the PCR reaction program is: performing pre-denaturation at 94 deg.C for 5min; 30s at 94 ℃;60 ℃ for 40s;72 ℃,1min45s,38 cycles; 72 ℃ for 10min.

The obtained PCR product was analyzed by agarose gel electrophoresis, and as shown in FIG. 1, a specific amplified band was observed between 2kb and 3kb under UV irradiation. The resulting extract was purified by agarose gel recovery kit (9672, takara).

Adding A into the purified DNA fragment by using a blunt end adding A reagent, connecting the DNA fragment with pMD20-T vector (6019, takara) through TA cloning, transforming the ligation product into Escherichia coli DH5a, picking 2-3 positive clones from an LB culture plate containing ampicillin (100 mg/L) for sequencing analysis, and the result shows that Lilium regoliumLrPAL-1The full-length gene sequence is shown in SEQ ID NO.1 and comprises an open reading frame (containing a termination code) of 2274 bp.

The protein sequence of Lilium regale LrPAL-1 obtained by DNAman software translation according to SEQ ID No.1 is shown in SEQ ID No.2 and contains 757 amino acids (a termination signal). On-line analysis by protein subcellular localization (Wo)LF PSORT) showed that the expression localization probability of LrPAL-1 in plant cells was chlo: 6, E.R.: 3, cyto: 1.5, cyto _ cycle: 1.5, mito: 1, plas: 1, and golg: 1, in this order. On-line prediction by cNLS Mapper showed that there is a nuclear localization signal sequence (VGSRIKCRTW) at the C-terminus of LrPAL-1 protein. The on-line analysis of ExPASy ProtParam showed that the molecular weight of LrPAL-1 was about 82.4kDa and the isoelectric point was 6.17. The full-length nucleotide sequence and amino acid sequence of LrPAL-1 were BLAST aligned in NCBI database, and found to be closest to the spliced sequence Lilium regale PAL (GenBank: KX842518.1 and ASV 46344.1) obtained by RNA sequencing, with a similarity of 95.07% (2162/2274) and 97.36% (737/757), respectively, and with a difference of 112 nucleotides and 20 amino acid sequences (amino acid sequence alignment is shown in FIG. 2). The amino acid sequence of Lilium regale LrPAL-1 has different degrees of similarity with other plant PAL amino acid sequences, such as: the similarity with wild banana PAL (XP _ 009417692.1) is 82.06 percent, the similarity with date PAL (XP _ 008791889.1) is 79.97 percent, the similarity with camphor tree PAL (RWR 79518.1) is 73.33 percent, and the similarity with musk lily LlPAL-1 (GQ 165724.1) is 54.46 percent, and the like. From the comparison resultLrPAL-1The gene is a novel gene and has no report.

Example 2 construction of plant overexpression vectors

(1) Construction of pART-CAM: (LrPAL-1) overexpression vector

Lilium regaleLrPAL-1Full-length gene sequence (SEQ ID NO. 1), primers LrPAL-1-inf-F and LrPAL-1-inf-R are designed, and seamless cloning (In-fusion) vector adaptor sequence is introduced into the primers. The positive cloning plasmid connected with TA in example 1 was used as a template, and LrPAL-1-inf-F and LrPAL-1-inf-R were used as primers to perform gene cloningLrPAL-1Specific amplification of (3).

The primer sequences are as follows:

LrPAL-1-inf-F: 5’-GGAGAGGACACGCTCGAGATGGCATCCAAGGACAGCT-3’

LrPAL-1-inf-R: 5’-TTAAAGCAGGACTCTAGATCAGAGATTCCAGTACCCCCT-3’

in which the In-fusion cloning vector linker sequence is underlined.

PCR amplification System: 2xfast pfu master Max 10ul, 1. Mu.L of forward primer (LrPAL-1-F, 10. Mu.M), 1. Mu.L of reverse primer (LrPAL-1-R, 10. Mu.M), 1. Mu.L of template (DNA), sterile ddH ₂ Make up to 20. Mu.L of O.

The PCR reaction program is: performing pre-denaturation at 94 deg.C for 5min; 30s at 94 ℃;60 ℃ for 40s;72 ℃,1min45s,38 cycles; 72 ℃ for 10min.

Will be provided withPAnd detecting the CR amplification product by agarose gel electrophoresis. The amplified target fragment has the same size as the expected fragment, and is recovered and purified according to the procedures of the gel recovery kit (9672, takara) instructions, thereby obtaining the target gene fragment.

The plant binary expression vector pART-CAM plasmid was digested with XbaI and XhoI. The enzyme digestion system is as follows: pART-CAM vector 1. Mu.g; xbaI 2. Mu.L; xhoI 1 μ L;10xTango buffer 4. Mu.L; sterile ddH ₂ O is complemented to 20 mu L; reaction at 30 ℃ for 1.5h, reaction at 37 ℃ for 1.5h, reaction at 65 ℃ for 20min. After the enzyme digestion, the vector large fragment is recovered according to a Takara agarose gel recovery kit.

Construction of pART-CAM:: lrPAL-1 overexpression vector was performed using the seamless Cloning technique (In-fusion HD Cloning Kit, takara).

The recombination reaction system is as follows:

Purifed PCR fragment (recovered)LrPAL-1Fragment of interest) 50ng; linear vector (pART-CAM) 100-150ng;5x In-fusion HD EnzymeP2 mu L of remix; sterile ddH ₂ Make up to 10. Mu.L of O. Mixing, and standing at 37 deg.C for 30 min. Then transforming the recombinant reaction system into Escherichia coli DH5a according to the molecular cloning experimental instruction, coating the Escherichia coli DH5a on LB culture medium containing streptomycin (50 mu g/mL), and obtaining correct content by screening and sequencing positive clonesLrPAL-1The recombinant expression vector pART-CAM of the gene segment is LrPAL-1. Target gene in recombinant expression vectorLrPAL-1The 5' end of (A) is located in a constitutive promoterP _35S Downstream, it enablesLrPAL-1Gene overexpression;LrPAL-1the 3' end of the fusion gene is provided with an OCS terminator, and the transcription of the fusion gene can be effectively terminated. On the recombinant expression vector is assemblednptIIGenes as transgenic plantsThe selection marker can be used for screening transgenic plants by using kanamycin. The assembly of LB and RB sequences on a recombinant expression vector, facilitating the assembly of an expression framework and a selectable marker gene therebetweennptIIIntegrated into the plant genome.

The Plasmid LrPAL-1, pART-CAM in the Escherichia coli is extracted and purified by a TIAnprep Mini Plasmid Kit Plasmid extraction Kit (DP 103, TIANGEN). The constructed pART-CAM is characterized in that the LrPAL-1 overexpression vector is transferred into agrobacterium EHA105 (AC 1010, shanghai Dingqing) competent cells by a liquid nitrogen freeze-thaw method, and positive clones are obtained, about 20 percent of glycerol is added and stored at-80 ℃ for later use.

Example 3 Agrobacterium-mediated transformation of tobacco and selection of transgenic Positive lines

Mixing tobacco (A)Nicotiana tabacumcv, petit Havana SR 1) seeds are washed clean with sterile water, sterilized with 75% ethanol for 1min, sterilized with sodium hypochlorite solution (2% available chlorine) for 15min, washed with sterile water for 3-5 times, and then inoculated on LS medium (PH 5.8) with sterile filter paper to remove water from the seed surface.

Glycerol bacteria were streaked on YEB solid medium (50. Mu.g/mL streptomycin + 20. Mu.g/mL rifampicin), after two days of culture at 28 ℃, single colonies were picked and inoculated into 5mL YEB liquid medium (50. Mu.g/mL streptomycin + 20. Mu.g/mL rifampicin), incubated overnight at 28 ℃ with shaking at 220rpm at constant temperature to OD ₆₀₀ When the concentration was about 0.6, the cells were collected by centrifugation at 5000rpm for 10min, and the collected cells were resuspended to OD with MS liquid medium ₆₀₀ =0.4。

Taking sterile and tender leaves after 1 to 2 months, removing edges and main veins, and cutting into small blocks of 0.5 x 0.5. Placing the cut explant in the resuspended Agrobacterium liquid, gently shaking for 5min, sucking the liquid on the surface of the explant with sterile filter paper, and placing the explant on a co-culture medium (MS +6-BA 1mg/L PH 5.8) for dark culture for 2-3 days. After co-cultivation, the explants were transferred to shoot-inducing differentiation medium (MS +6-BA 1mg/L + Timentin 300mg/L + Kan 100mg/L pH 5.8). Sprouting was induced and the medium was changed every two weeks.

When the resistant bud grows to 2cm, cutting the bud, transferring the bud into a rooting culture medium (1/2MS + Timentin 300mg/L + IAA 0.1mg/L + Kan 100mg/L PH 5.8), cutting part of leaves when a complete plantlet is formed, extracting genome DNA by adopting a CTAB method, and detecting a positive plant by using gene specific primers (35S-F1 and LrPAL-1-R1).

35S-F1：5'-TGACGCACAATCCCACTATC-3'

LrPAL-1-R1：5'-GTGAGAAGTCCGGCGATATAC-3'

The PCR amplification system was 2 × TransDirect PCR SuperMix (+ dye) 10ul, forward primer (35S-F, 10. Mu.M) 1. Mu.L, reverse primer (LrPAL-1-R2, 10. Mu.M) 1. Mu.L, template (DNA) 1. Mu.L, sterile ddH ₂ Make up to 20. Mu.L of O.

The PCR reaction program is: pre-denaturation at 94 deg.C for 5min; 30s at 94 ℃; at 62 ℃ for 40s; 45s,38 cycles at 72 ℃;72 ℃ for 10min.

The PCR detection result showed (FIG. 3), the target fragment could not be detected in the wild type plant (WT), and only the target fragment (about 776 bp) was amplified in the transgenic plant and the positive control, indicating that the exogenous gene was presentLrPAL-1Has been introduced into the tobacco genome. The total T0 generation positive plants obtained in the test are 9 plants which are respectively T0-1, T0-3, T0-5, T0-7, T0-9, T0-11, T0-12, T0-14 and T0-16.

Example 4: phenotypic observation and fungal resistance analysis of transgenic tobacco

And (4) collecting seeds from the positive plants of the T0 generation, and continuously sowing to obtain transgenic tobacco strains of the T1 generation. Total RNA was extracted from wild-type and transgenic tobacco using TaKaRa MiniBEST Plant RNA Extraction Kit (9769, takara) and PrimeScript was used ^TM Reverse transcription of RT reagent Kit with gDNA Eraser (PR 047A, taKaRa) to obtain cDNA, and extracting with tobaccoActinThe gene is used as an internal reference and SYBR Premix Ex Taq is used ^TM II (PR 820A, taKaRa) reagent qRT-PCR detection, analysis of transgenic Positive lines and wild type tobaccoLrPAL-1The level of gene expression.

Detection ofActinThe gene primers are as follows:

Actin-qF: 5'- AATGGAACTGGAATGGTCAAGGC-3'

Actin-qR:5'- TGCCAGATCTTCTCCATGTCATCCCA-3'

detection ofLrPAL-1The gene primers are as follows:

LrPAL-1-qF：5'-GTGACTACTACAATGACGGCT-3'

LrPAL-1-qR：5'- TCCATCGCCTGGCACAGACC-3'

the results are shown in FIG. 4A, in comparison with wild type plants (WT), in 3 representative transgenic lines: (A), (B), (C)OX-L1、OX-L7AndOX-L9) Middle-purpose gene Lilium regaleLrPAL-1Are all heterologously up-regulated, andOX-L7the expression level was highest and could not be detected in wild type plants (WT)LrPAL-1Is expressed to show thatLrPAL-1Has been introduced into the tobacco genome and successfully transcribed.

For further confirming the transgenic line, a conventional Phenylalanine Ammonia Lyase (PAL) activity measuring method is adopted, 3 g of wild type and transgenic tobacco leaf samples are taken, 10 ml of 0.1 mol/L boric acid buffer solution (PH =8.8, containing 5 mmol/L mercaptoethanol) and 0.3 g of polyvinylpyrrolidone (PVP) are added, a small amount of quartz sand is ground into a mortar to be homogenized, the homogenized slurry is centrifuged for 15min at 10000 r/min, and the supernatant is taken to be constant volume to 10 ml for measuring the enzyme activity. 1 ml of supernatant, 1 ml of phenylalanine and 2ml of distilled water were taken, and the blank was replaced with 1 ml of a mercaptoethanol-containing boric acid buffer without adding a substrate. Placing in 30 deg.C constant temperature water bath, keeping the temperature for 60 min, adjusting to zero with contrast, and measuring absorbance with spectrophotometer at wavelength of 290nm. To measure the tube reaction solution A per hour ₂₉₀ The increase of 0.01 is one enzyme activity unit (U). The results show (FIG. 4B), 3 representative transgenic lines: (FIG. 4B)OX-L1、OX- L7AndOX-L9) The activity of the medium PAL enzyme is obviously higher than that of wild tobacco, whereinOX-L7The PAL activity of the enzyme is highest.

Phenotypic observation of tobacco plants normally cultured in a greenhouse for 2 months is shown in fig. 5 and 6, and the transgenic tobacco leaves show obvious hyperplasia and wrinkling phenomena and are obviously different from wild type.

Respectively taking the cross section of the 3 rd node of wild tobacco and transgenic tobacco by conventional biochemical method, and referring to the formula of Lisuper Feng (functional research of Lisuper Feng, poplar transcription factors PtoMYB128 and PtoMYB74 in secondary wall biosynthesis, doctor's treatise on university of Chinese academy of sciences, 12 months 2018)The method utilizes phloroglucinol dye liquor to carry out lignin dyeing analysis, and the result shows that (figure 7) 3 representative transgenic lines (figure 7)OX-L1、OX-L7AndOX-L9) Compared to wild type plants (WT), significantly increased in the degree of lignification in the stem. It can be seen that there is over-expressionLrPAL-1The gene promotes the synthesis and accumulation of lignin in tobacco stems, and further can increase the lodging resistance of tobacco.

The wild type and the transgenic tobacco in vitro leaves at the same position are respectively taken and evenly placed in a culture dish by adopting a conventional biochemical method. Pricking 2-3 micro wounds at the same position of the leaf with a needle tip, taking pathogenic bacteria with the same size from a botrytis cinerea plate cultured for about 1 week by using a puncher, directly inoculating the pathogenic bacteria to the wound of the leaf, preserving moisture, and standing at the normal temperature of 25 ℃ for about 3 days. As a result, it was found (FIG. 8A, FIG. 8B) that the leaves of the 3 representative transgenic lines were less or hardly damaged by Botrytis cinerea, compared to the wild-type tobacco leaves, while the leaves of the wild-type tobacco leaves were infected by Botrytis cinerea over a large area. Similarly, the wild type and the transgenic tobacco in vitro leaves at the same position are uniformly placed in a culture dish. Pricking 2-3 micro wounds at the same position of the leaf with a needle tip, taking pathogenic bacteria with the same size from an alternaria alternate plate cultured for about 1 week by using a puncher, directly inoculating the pathogenic bacteria to the wound of the leaf, preserving moisture, and standing at the normal temperature of 25 ℃ for about 7 days. The results show (FIG. 9A) that the leaves of 3 representative transgenic lines were less infected by Alternaria alternata, whereas the leaves of wild type tobacco were more infected by Alternaria alternata. The area of infection by Alternaria alternata in wild-type tobacco leaves was about 4 times that of the leaves of the transgenic line (FIG. 9B). Taken together, the results indicate that overexpressionLrPAL-1The gene improves the resistance of the transgenic tobacco to pathogenic bacteria such as botrytis cinerea and alternaria.

In addition, referring to the method of Tengtiantong and the like (Tengtiantong, zhanxiaolong, gongkun and the like, 2015, 34 (7): 744-750) for researching the process of extracting the total flavonoids in the pomegranate leaves by optimizing ultrasonic-microwave synergy on response surfaces, 1 g of wild type and transgenic tobacco leaves are respectively taken, fully ground by liquid nitrogen, and extracted for 24 hours after the volume is fixed to 10 ml by 75 percent ethanol; taking 1 ml of extracting solution, and respectively adding NaNO with the mass fraction of 5 percent ₂ Solution 0.3ml, shaking, standing for 6 min, adding 10% Al (NO) ₃ ） ₃ 0.3 ml of solution is shaken up, placed for 6 min and then added with 4 ml of NaOH solution with the mass fraction of 4 percent, finally 75 percent ethanol is used for fixing the volume to 10 ml, shaken up, placed for 12 min, the light absorption value is measured at 508 nm, according to the linear regression equation A =11.201C-0.013 ² =0.9998 converted to the flavone content in the sample. The results are shown in FIG. 10, althoughOX-L9The content of total flavonoids in the leaf of the transgenic tobacco line is slightly increased, and the total flavonoids content in the leaf of the transgenic tobacco line is not obviously different from that in the Wild Type (WT) in general, which indicates that the LrPAL-1 is not involved in the regulation of flavonoid pathways.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

SEQUENCE LISTING

<110> Changjiang university academy;

<120> Lilium regale LrPAL-1 gene and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2274

<212> DNA

<213> Lilium regale Wilson

<400> 1

atggcatcca aggacagctc ccccgcccag gccatcaccc gagccctaaa ggacttccgt 60

cccccaaccc tcccagtccc cgtcaacggc atcaacccca tcgccgtctc cggctcctcc 120

tacgtcccca accctcctca ctggaagaag gccgccgagg ccctcagctg caaccacttc 180

gacgaagtcc accgcatggt cacccagttc accaccctct ccactgtcga actccagggc 240

actactctca cagtcgccca agtcgctgcc atcgcccacc gcacttccgt tgccgttgcc 300

cttgacgagc ctacggccag ggcacgtgtc gaccgcagcg ctcgctgggt gtccgacaac 360

atcgagcgcg gcaccgacac ctacggtgtc accaccggct tcggcgccac atctcaccgc 420

cggacaaaga agactgccga tctccaagcc gagcttatcc ggttcctgaa tgccggtgtg 480

atcggaaagg agtcgctgcc gacgagctac gccaagatcg cgatgctggt caggactaac 540

actctgatgc aggggtactc ggggattcga tgggagcttc tcgaggccat ggctactctc 600

atgaaccaga atctcatacc gaagttgccg ctccgaggta ccatctcggc gtctggcgat 660

ctcgtgccgc tgtcgtatat cgccggactt ctcaccggcc ggcacaactc caaggtcacc 720

accccagaag gcggggtcat cacctccact gaggctttga agcgtgctgg gatcaatgcc 780

ccctttgaac tccaggcaaa ggagggccta gctctcgtca acggcacggt cgtcggctcg 840

gcagtcgcag ccaccgtctg ctacgactcc aatatcctcg ctctcctctc cgttgtcctc 900

tccgccatgt tctgtgaggc aatgcagggg aagccggagt tcaccgaccc cctcacccac 960

gagctcaagc accaccccgg ccaaatcgaa tccgctgcca ttatggagta cctcctcgaa 1020

ggcagcgact acatgcgcga agctcgcctc cgccacgagc gtgagccttt gaccaagcca 1080

aagcaggacc gctacgccct ccgaacatcc ccacaatggc ttgggcctca aatcgaggtt 1140

atccgtgctg ccacccacgc catcgagcgc gagatcaact ccgtcaacga caacccactc 1200

atcgatgtcg cccgcgacat cgccctgcac ggtggaaatt ttcagggcac gcctattggc 1260

gtttcgatgg ataccctccg aatatccttg gctgccattg ggaagcttgt gtttgctcaa 1320

ttctcagagt tggtatgtga ctactacaat gacggcttgc cgtcaaacct cagcgctgga 1380

gccgacccca gcctcgatta cggcctgaaa ggggcggaga ttgcgatggc cagttactgc 1440

tcggagctcc agtaccttgc taacccggtg acaacacatg ttcagagcgc agagcaacac 1500

aatcaggatg tgaactcgct agggttgata tctgcacgga agtcggctga ggcggtggag 1560

atactgaagc tgatgatgtc aacgttcatg gttggtctgt gccaggcgat ggacctgagg 1620

cagatggagg agaatttgcg agaggttgtg aaacatgcgg tggttgcggc tgcggagatc 1680

acggagttga gtgctaatgc tgatgttttg aaggagttgg tgcaggttgt agagaggcag 1740

ccggtgtatt cttacattga tgatccggcg aacccggcat atgcactaat gctgcagctg 1800

agggaggtag tggtggagcg ggcaatcggt gggtcggaag aggggatggc agccttcaag 1860

aaggtgccag cttttcaggc tgagttgaag gagaggctta acaaggaggt gggaagggcg 1920

agagagaggt ttgggagagg ggattttgtt gtgggaagta ggataaagaa atgcaggaca 1980

tggcctgtgt acaagctggt gagtgaggag gtggggacgg agcttctgac gggggagaag 2040

aaggtgagcc ccggagagtt tatcgagaag gtgtatgagg aggtgatggg ggatagtggg 2100

aagatagggg tggtgctggt cgggtgtctg gcaggatgga ggggggcggc agggccgttc 2160

acgccacggc cggaggtgtc gtcaccagcg tacaaaaatc ctgagacttg gggttggttc 2220

gatcaggcaa ggtctccatc ggctaccagt ggaagggggt actggaatct ctga 2274

<210> 2

<211> 757

<212> PRT

<213> Lilium regale Wilson

<400> 2

MASKDSSPAQ AITRALKDFR PPTLPVPVNG INPIAVSGSS YVPNPPHWKK AAEALSCNHF 60

DEVHRMVTQF TTLSTVELQG TTLTVAQVAA IAHRTSVAVA LDEPTARARV DRSARWVSDN 120

IERGTDTYGV TTGFGATSHR RTKKTADLQA ELIRFLNAGV IGKESLPTSY AKIAMLVRTN 180

TLMQGYSGIR WELLEAMATL MNQNLIPKLP LRGTISASGD LVPLSYIAGL LTGRHNSKVT 240

TPEGGVITST EALKRAGINA PFELQAKEGL ALVNGTVVGS AVAATVCYDS NILALLSVVL 300

SAMFCEAMQG KPEFTDPLTH ELKHHPGQIE SAAIMEYLLE GSDYMREARL RHEREPLTKP 360

KQDRYALRTS PQWLGPQIEV IRAATHAIER EINSVNDNPL IDVARDIALH GGNFQGTPIG 420

VSMDTLRISL AAIGKLVFAQ FSELVCDYYN DGLPSNLSAG ADPSLDYGLK GAEIAMASYC 480

SELQYLANPV TTHVQSAEQH NQDVNSLGLI SARKSAEAVE ILKLMMSTFM VGLCQAMDLR 540

QMEENLREVV KHAVVAAAEI TELSANADVL KELVQVVERQ PVYSYIDDPA NPAYALMLQL 600

REVVVERAIG GSEEGMAAFK KVPAFQAELK ERLNKEVGRA RERFGRGDFV VGSRIKKCRT 660

WPVYKLVSEE VGTELLTGEK KVSPGEFIEK VYEEVMGDSG KIGVVLVGCL AGWRGAAGPF 720

TPRPEVSSPA YKNPETWGWF DQARSPSATS GRGYWNL 757

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<400> 3

atggcatcca aggacagct 19

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

tcagagattc cagtaccccc t 21

<210> 5

<211> 37

<212> DNA

<213> Artificial sequence

<400> 5

ggagaggaca cgctcgagat ggcatccaag gacagct 37

<210> 6

<211> 39

<212> DNA

<213> Artificial sequence

<400> 6

ttaaagcagg actctagatc agagattcca gtaccccct 39

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

tgacgcacaa tcccactatc 20

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

gtgagaagtc cggcgatata c 21

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<400> 9

aatggaactg gaatggtcaa ggc 23

<210> 10

<211> 26

<212> DNA

<213> Artificial sequence

<400> 10

tgccagatct tctccatgtc atccca 26

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<400> 11

gtgactacta caatgacggc t 21

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<400> 12

tccatcgcct ggcacagacc 20

Claims (4)

1. Lilium regaleLrPAL-1Application of gene in inhibiting growth of Botrytis cinerea and/or Alternaria alternata, and is characterized in that the Lilium regaleLrPAL-1The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2.

2. Lilium regaleLrPAL-1The application of the gene in promoting the proliferation and wrinkling of plant leaves is characterized in that the Lilium regaleLrPAL-1The gene is applied to tobacco; lilium regale (Lilium regale L.) MillLrPAL-1The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2.

3. Lilium regaleLrPAL-1The application of the gene in regulating and controlling the lodging resistance of plants is characterized in that the Lilium regaleLrPAL-1The gene is applied to tobacco; lilium regaleLrPAL-1The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2.

4. A method of producing a transgenic plant comprising the steps of: introduction of Lilium regale into recipient plantsLrPAL-1Gene, obtaining transgenic plants; lilium regaleLrPAL-1The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the gene is shown as SEQ ID NO. 2; the transgenic plant has enhanced disease resistance or enhanced lodging resistance or altered leaf morphology as compared to the recipient plant; the plant is tobacco.

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