CHLORELLA VARIABILIS-DERIVED PHOSPHOMANNOSE ISOMERASE GENE AND APPLICATION THEREOF

Abstract

The present invention provides a Chlorella variabilis-derived phosphomannose isomerase gene, herein named ChloPMI. The present invention also provides a prokaryotic expression vector comprising ChloPMI, which can be used for identifying mannose metabolic activity of ChloPMI protein. Further, the present invention provides an expression cassette and a plant expression vector comprising ChloPMI, and a use of the expression cassette and the expression vector in genetic transformation of plants. According to the present invention, the transformation of rice cells is successfully achieved with the plant expression vector constructed from the ChloPMI gene using mannose as a selection agent. According to the present invention, a plant-derived phosphomannose isomerase gene is successfully separated and cloned from Chlorella variabilis. Since the plant-derived phosphomannose isomerase gene is derived from Chlorella variabilis, it is environment-friendly and has no potential hazard to human, which is very beneficial in promoting and applying transgenic products and eliminating any existing doubts on transgenes.

Claims

1. A phosphomannose isomerase gene from Chlorella variabilis having a nucleotide sequence represented by SEQ ID NO: 1.

2. A prokaryotic expression vector comprising the phosphomannose isomerase gene according to claim 1.

3. A prokaryotic identification method for identifying mannose metabolic activity of a phosphomannose isomerase gene, comprising: performing color identification on an expression strain comprising the phosphomannose isomerase gene according to claim 1 by a color identification method with phenol red.

4. An expression cassette comprising the phosphomannose isomerase gene according to claim 1.

5. A plant expression vector comprising the phosphomannose isomerase gene according to claim 1.

6. A method for obtaining transformed rice cells by a mannose-based selection with a plant expression vector pCAMBIA1381-ChloPMI comprising the phosphomannose isomerase gene according to claim 1, comprising the steps of: (1) separating embryos from sterilized rice seeds with shell removed and placing the embryos on callus induction medium to generate secondary callus; (2) transferring the secondary callus to new callus induction medium for pre-culture to obtain the callus for the transformation; (3) contacting the callus obtained in the step (2) with Agrobacterium for 15 min, wherein the Agrobacterium is incorporated with the plant expression vector carrying the phosphomannose isomerase gene; (4) transferring the callus treated in the step (3) into a culture dish lined with a sterile filter paper, and culturing for 48 h at 21-23° C.; (5) placing the callus treated in the step (4) on a pre-selection medium, and culturing for 5-7 days; and (6) transferring the callus treated in the step (5) onto a selection medium to obtain resistant callus, that is, transformed rice cells which can metabolize mannose.

7. A use of the phosphomannose isomerase gene, the expression cassette and the plant expression vector, the phosphomannose isomerase gene from Chlorella variabilis having a nucleotide sequence represented by SEQ ID NO: 1, the expression cassette comprising the phosphomannose isomerase gene, the plant expression vector comprising the phosphomannose isomerase gene or the expression cassette, wherein transformed plant cells are obtained by the method according to claim 6 using the phosphomannose isomerase gene as a selection marker, and the resulting transformed plant cells are used to obtain a transgenic plant or plant part.

8. The use according to claim 7, wherein the plant comprises a cereal crop, vegetable crop, flower crop, and energy crop.

9. The use according to claim 7, wherein the plant part comprises a cell, protoplast, cell and tissue culture, callus, cell mass, plumule, pollen, ovule, petal, style, stamen, leaf, root, root tip, anther, and seed.

10. A prokaryotic identification method for identifying mannose metabolic activity of a phosphomannose isomerase gene, comprising: performing color identification on an expression strain comprising the prokaryotic expression vector according to claim 2 by a color identification method with phenol red.

11. A plant expression vector comprising the expression cassette according to claim 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows a schematic illustration of a pCAMBIA1381-ChloPMI vector plasmid constructed using the phosphomannose isomerase gene of the present invention.

[0029] FIG. 2 shows a picture of identifying ChloPMI activity through a staining reaction, where NC (negative control) is E. coli expressing strain BL21 transformed with a pGEX-6P-1 empty vector; 1 is BL21 strain containing Chlorella variabilis ChloPMI expression vector pGEX-ChloPMI; and 2 is BL21 strain containing E. coli PMI expression vector pGEX-PMI.

[0030] FIG. 3 shows a picture of resistant rice callus produced by a mannose-based selection after transformation with Agrobacterium containing a pCAMBIA1381-ChloPMI plasmid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Unless otherwise specified, the operations in specific embodiments described below are performed following general conventional operations in the art. A person skilled in the art can easily obtain the teaching regarding such conventional operations from the prior art, for example, can make reference to the textbook, Sambrook and David Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Vols 1, 2; Charles Neal Stewart, Alisher Touraev, Vitaly Citovsky and Tzvi Tzfira, Plant Transformation Technologies and the like. Unless specifically stated, medicinal raw materials and reagent materials used in the examples below are commercially available products.

[0032] Specific embodiments of the present invention will be described below in detail with reference to the accompanying drawings. It should be noted that the figures of experimental results illustrated in the drawings are originally colored diagrams; however, in view of the provisions in the patent law, the applicant converts them to grayscale images, and despite this, the difference between the experimental results under different conditions still can be distinguished from shades in the images.

EXAMPLE 1

Obtaining and Cloning a ChloPMI Gene

[0033] A sequence with the highest homology, namely, the ChloPMI protein sequence, was obtained by aligning homologous sequences in the genomic sequence (genome.jgi-psf.org) of Chlorella variabilis which can utilize mannose based on phosphomannose isomerase protein sequence of the bacteria. The phosphomannose isomerase protein sequence of the bacteria is as follows:

TABLE-US-00003 MQKLINSVQNYAWGSKTALTELYGMENPSSQPMAELWMGAHPKSSSR VQNAAGDIVSLRDVIESDKSTLLGEAVAKRFGELPFLFKVLCAAQPL SIQVHPNKHNSEIGFAKENAAGIPMDAAERNYKDPNHKPELVFALTP FLAMNAFREFSEIVSLLQPVAGAHPAIAHFLQQPDAERLSELFASLL NMQGEEKSRALAILKSALDSQQGEPWQTIRLISEFYPEDSGLFSPLL LNVVKLNPGEAMFLFAETPHAYLQGVALEVMANSDNLRAGLTPKYID IPELVANVKFEAKPANQLLTQPVKQGAELDFPIPVDDFAFSLHDLSD KETTISQQSAAILFCVEGDATLWKGSQQLQLKPGESAFIAANESPVT VKGHGRLARVYNKL

[0034] Thereafter, RNA of Chlorella variabilis was further extracted and reverse transcribed into cDNA. According to the coding sequence (CDS) of ChloPMI, gene specific cloning primers, a forward primer 5′-ATGGCTGGAACGGCGACAGAGA-3′ and a reverse primer 5′-TCACTCAAAGGCCATTCCGTTG-3′ were designed, and then PCR amplification was performed using the cDNA as a template.

[0035] PCR-amplified target fragments having a fragment length of 1278 bp were recovered, and were ligated to PGEM-T-Easy vector (available from Promega Inc.) according to the instructions for the vector. After the transformation of E. coli competent cells XL-Blue using thermal stimulation, positive clones were obtained via colony PCR screening. The identified positive clones were delivered to Invitrogen Inc. for sequencing. The correct clones by verification were recombinant plasmids containing ChloPMI, named as PGEM-T-ChloPMI. The nucleotide sequence of ChloPMI was represented by SEQ ID NO: 1.

EXAMPLE 2

Construction of a Prokaryotic Expression Vector Containing the ChloPMI Gene

[0036] PCR amplification was performed by the design of ChloPMI prokaryotic expression primers, a forward primer 5′-GGATCC ATGGCTGGAACGGCGACAGAGA -3′ (underline indicates BamHI restriction site) and a reverse primer 5′-CTCGAGCTCAAAGGCCATTCCGTTG-3′ (underline indicates XhoI restriction site) using the PGEM-T-ChloPMI recombinant plasmid as a template. The prokaryotic expression vector pGEX-ChloPMI fused to GST (glutathione-S-transferase) fragment was then obtained by ligating the recovered PCR-amplified target fragments to pGEX-6P-1 expression vector (available from GE Inc.) digested with BamHI and XhoI, and was transformed into E. coli expressing strain BL21. Simultaneously, a pGEX-6P-1 empty vector and pGEX-PMI containing E. coli phosphomannose isomerase expression vector were also transformed into E. coli expressing strain BL21, respectively.

EXAMPLE 3

ChloPMI Activity Analysis

[0037] The BL21 strain containing the prokaryotic expression vector pGEX-ChloPMI, pGEX-PMI and pGEX-6P-1 empty vector was streaked. Monoclones were picked and inoculated into LB liquid medium (see Table 1 for composition, Agrobacterium culture medium, no agar), and cultured overnight with shaking at 37° C. (200 r/min). On the next day, the culture was centrifuged for 1 min at 6000 r/min at room temperature, the supernatant was discarded and the pellet was resuspended with a small amount of sterilized water. The resuspension was taken and plated on sterilized phenol red chromogenic medium (1% peptone, 0.5% NaCl, 50 mg/L phenol red, 30% mannose, PH 7.4) with 1:50. After 48 h, the change in medium color was observed. If the strain has an ability to metabolize mannose, the medium was acidified, resulting in a drop in PH value. The medium color therefore gradually changed into yellow from red at PH 7.4. Results from activity analysis of different vectors were seen in FIG. 2. As can be seen from the figure, NC strain (E. coli expressing strain BL21 transformed with the pGEX-6P-1 empty vector) does not have the ability to metabolize mannose, as the medium color is still red. In contract, E. coli of 1 (BL21 strain containing Chlorella variabilis ChloPMI expression vector pGEX-ChloPMI) and 2 (BL21 strain containing E. coli PMI expression vector pGEX-PMI) have an ability to metabolize mannose, such that the medium is acidified, resulting in a drop in PH value, and the color in medium thus gradually changes into yellow from red at PH 7.4.

EXAMPLE 4

Construction of a ChloPMI Plant Expression Vector

[0038] PCR amplification was performed by a forward primer 5′-CTCGAGATGGCTGGAACGGCGACAGAGA -3′ (underline indicates XhoI restriction site) and a reverse primer 5′-CTCGAGTCACTCAAAGGCCATTCCGTTG-3′ (underline indicates XhoI restriction site) using the PGEM-T-ChloPMI recombinant plasmid as a template. The plant expression vector pCAMBIA1381-ChloPMl was then obtained by ligating the recovered PCR-amplified target fragments to pCAMBIA1381 vector linearized with XhoI via T.sub.4 ligase (FIG. 2). The plant expression vector was transformed into Agrobacterium tumefaciens strain EHA105 (deposited with Rice Research Institute, Anhui Academy of Agricultural Sciences) using the freeze-thaw method for genetic transformation.

Example 5

Genetic Transformation in Rice using ChloPMI as a Selective Marker Gene

[0039] 1. Induction and Pre-Culture of Mature Embryo Callus

[0040] Mature seeds of Nipponbare (deposited with Rice Research Institute, Anhui Academy of Agricultural Sciences) were peeled. The normal appearance, clean and free of mildew seeds were selected and shaken for 90 sec in the presence of 70% alcohol, and then, the alcohol was poured off. The seeds were further washed with 50% sodium hypochlorite solution containing Tween20 (available chlorine concentration of stock solution >4%, 1 drop Tween20 per 100 ml), and shaken for 45 min (180 r/min) on a shaking table. Sodium hypochlorite was poured off. The seeds were washed 5-10 times with sterilized water until no odour of sodium hypochlorite. Finally, the seeds were added with sterilized water, and soaked overnight at 30° C. Embryos were separated along aleurone layer with a surgical blade, placed with scutellum up on induction medium (see Table 1 for composition) at 12 embryos/dish, and cultured in the dark at 30° C. to induce the callus.

[0041] Two weeks later, spherical, rough, pale yellow secondary callus appeared. Pre-culture operation could be performed, that is, the secondary callus were transferred onto a new callus induction medium, and pre-cultured in the dark at 30° C. for 5 d. After the completion of the pre-culture, small particles having a good condition and a strong split were collected with a spoon into a 50 mL sterile centrifuge tube for Agrobacterium infection.

[0042] 2. Culture of Agrobacterium Strains and Preparation of Suspensions

[0043] Agrobacterium strain EHA105 (deposited with Rice Research Institute, Anhui Academy of Agricultural Sciences) containing pCAMBIA1381-ChloPMI vector was streaked on an LB plate containing 50 mg/L kanamycin (see Table 1 for composition), and cultured in the dark at 28° C. 24 h later, the activated Agrobacterium was inoculated with a sterile inoculation loop onto a fresh LB plate containing 50 mg/L kanamycin for a second activation, and cultured overnight in the dark at 28° C. 20-30 mL of Agrobacterium suspension medium (see Table 1 for composition) was added in a 50 mL sterile centrifuge tube. The Agrobacterium activated twice was scraped off by the inoculation loop, with optical density 660 nm (OD660) adjusted to about 0.10-0.25, and allowed to stand for over 30 min at room temperature.

[0044] 3. Infection and Co-Culture

[0045] The prepared callus (see step 1) were added with the Agrobacterium suspension, and soaked for 15 min with gently shaking from time to time. After the completion of soaking, the liquid was poured off (no liquid drops if possible). The excess Agrobacterium suspension on the callus surface was wicked away with sterile filter papers, and blow dried by sterile wind on an ultra-clean station. Three sterile filter papers were placed on a 100×25 mm disposable sterile culture dish, and 2.5 mL of the Agrobacterium suspension medium was added. The blotted callus were uniformly dispersed onto the filter papers, and cultured for 48 h in the dark at 23° C.

[0046] 4. Pre-Screening and Screening Culture

[0047] After the completion of the co-culture, the co-cultured callus were uniformly dispersed in a pre-selection medium (see Table 1 for composition), and cultured for 5 d in the dark at 30° C. After the completion of the pre-screening culture, the callus were transferred onto a selection medium (see Table 1 for composition) at 25 callus/culture dish, and cultured in the dark at 30° C. 2-3 weeks later, the growth of resistant calli is evident (as shown in FIG. 3), and differentiated regeneration could be performed. As can be seen in FIG. 3, the newborn resistant callus assume a pale yellow color, compact texture and strong granular sensation, indicating that embryonic callus have a better state, and are suitable for subsequent differentiation and regeneration. It should be noted that the figures of experimental results illustrated in the drawings are originally colored diagrams; however, in view of the provisions in the patent law, the applicant converts them to grayscale images, and despite this, the difference between the experimental results under different conditions still can be distinguished from shades in the images.

[0048] Using the resulting resistant callus, a rice plant or plant part can be cultured.

[0049] It will be understood that the specific embodiments described herein are merely used for illustrative purposes to help a person skilled in the art to better understand the invention, and are not intended to limit the scope of the invention. Without departing from the spirit and scope of the invention, various changes or variations which may be made by a person skilled in the art according to the invention are intended to be within the scope of appended claims.