CN104087598A - Maize calcineurin B albuminoid ZmCBL10 gene and applications thereof - Google Patents

Abstract

The invention provides a maize calcineurin B albuminoid ZmCBL10 gene. The coding amino acid sequence of the gene is shown as SEQ ID No.1. The nucleotide sequence of a coding sequence of the gene is shown as SEQ ID No.2. The ZmCBL10 gene participates in plant salt tolerance processes through gene regulation and control, and has significant importance for culturing of high-output genetically modified crops.

Description

Corn calcium adjusting phosphatase B analogy albumen ZmCBL10 gene and application thereof

Technical field

The present invention relates to genetically engineered field, specifically, relate to corn calcium adjusting phosphatase B analogy albumen ZmCBL10 gene and application thereof.

Background technology

Plant-growth is among the environment constantly changing, and environment is the important factor that affects growth and development of plants, and wherein adverse circumstance is coercing of must facing of plant.Plant is for perception and adapt to the finely regulating mechanism of adverse circumstance perception, signal conduction and stress response that environment stress develops a set of complexity on molecule, cell and physiological level.And numerous research shows, some abiotic stress and biology are coerced caused biochemical reactions all and Ca ²⁺signal transduction is relevant.Wherein, Ca ²⁺the variation of concentration on space-time represented the stress signal that certain is special, is referred to as calcium signal; And can perception Ca ²⁺the albumen of this change in concentration is referred to as calcium ion susceptor.

Ca ²⁺susceptor is at monitoring Ca ²⁺when concentration instantaneous variation and Ca ²⁺in conjunction with and occurred conformation on variation, thereby and variation in this conformation can be carried out the transmission of signal with its downstream interactions between protein.At present, the comparatively extensive and deep Ca of research in higher plant ²⁺susceptor mainly comprises three classes: calcium dependent protein kinases (CDPK), calmodulin (CaM) and calcium adjusting phosphatase B analogy albumen (CBL).What wherein research was comparatively deep is CDPK family, and the albumen of this family both can be in conjunction with Ca ²⁺, there is again protein kinase activity; With Ca ²⁺in conjunction with in conception at once change, realize its function by regulating and controlling effect in self molecule.And two other calcium ion susceptor, CaM and CBL do not possess enzymic activity, they experience Calcium Signal change after need to have an effect and signal could be handed on the protein kinase in downstream.Therefore, not only can will be able to be referred to as response type susceptor (sensor responders) in conjunction with calcium ion but also this class susceptor with kinase activity, and the calcium ion susceptor that can only there is no in conjunction with calcium ion kinase activity on the other side is called dependent form susceptor (sensor relays).

In recent years, the research of CBL family is risen gradually, CBL contains 4 can be directly in conjunction with Ca ²⁺typical EF-hand structural domain, in conjunction with after calcium ion activation, can be combined with a class Special Proteins kinases in downstream, and activate its activity.This class can be called CBL interact protein kinases (CBL-interacting protein kinases, CIPKs) with the kinases of CBL specific combination.CIPK contains serine/threonine protein kitase district at N end, and does mutually with CBL by its distinctive C-terminal region (C-terminal region).By the scanning analysis discovery of the genomic data to known (comprising EST), CBL and CIPK are only present in plant, illustrate that they may be the peculiar genoid families of plant, may come across in the early stage evolution of plant.

Research shows, has all found the existence of CBLs gene family in many plants; Wherein, in Arabidopis thaliana, paddy rice, corn and willow, find 10 CBLs, in Chinese sorghum and grape, find respectively 6 and 8 CBLs genes.In recent years the research of CBL gene family is mainly concentrated in model plant Arabidopis thaliana, and the CBL gene of having reported participates in the response of Arabidopis thaliana to environment stress mostly.AtCBL4/SOS3 is the gene that plays critical function in the time of Arabidopis thaliana reply salt stress of reporting the earliest, and this gene is mainly expressed and done mutually with AtCIPK24/SOS2 in root, the latter can phosphorylation NHX7/SOS1 by Na ⁺transport cell, reduce Na in body ⁺content; AtCBL10/SCaBP8 mainly expresses in stem and blade, has participated in Na ⁺separating, reduces the infringement of high salt pair plant; AtCBL1 and AtCBL10 by participate in different signal paths to plant response adverse circumstance especially to K ⁺and NO ₃ ^-absorption process in play an important role.AtCBL2 and AtCBL3 can be by light inductions, and in ionic equilibrium, play an important role maintaining in cell.In addition AtCBL5 also plays an important role in the high salt of reply and osmotic stress.The function of AtCBL6, AtCBL7 and AtCBL8 does not have report.Only find that for the research of corn C BLs gene ZmCBL4 plays an important role in reply high-salt stress.

Therefore, for a research field in the ascendant just of plant CBL gene, only results of study are the desk study to this field just at present, and the research work of this respect is carried out substantially on model plant Arabidopis thaliana.Research for this genoid of important crops (as paddy rice, corn, wheat etc.) also rarely has report, particularly about the functional study of corn C BL gene there is not yet application.

Summary of the invention

In order to solve problems of the prior art, the object of this invention is to provide a kind of corn calcium adjusting phosphatase B analogy albumen ZmCBL10 gene and application thereof.

In order to realize the object of the invention, first the present invention provides a kind of corn calcium adjusting phosphatase B analogy albumen ZmCBL10 gene, and the aminoacid sequence of its coding is as shown in SEQ ID No.1.

As preferably, the nucleotide sequence of the encoding sequence of described gene is as shown in SEQ ID No.2.

The present invention also provides the primer pair for the ZmCBL10 gene coded sequence that increases, and described primer pair comprises:

Forward primer F:5'-atgccacgagccactgaggcggctt-3';

Reverse primer R:5'-ctaatcttccaccgccgtgttg-3'.

The present invention also provides the carrier that contains aforementioned gene.

The present invention also provides the engineering bacteria that contains aforementioned gene.

The present invention also provides the application of aforementioned gene in improvement plant anti-salt is coerced.

Further, aforementioned gene is transformed in plant plant by carrier or engineering bacteria, obtains transgenic plant plant, thus the resistance that improvement plant salt is coerced.

Be specially:

1) taking corn cDNA as template, utilize aforementioned primer pair amplification ZmCBL10 gene order, after adding HA label, sequence end is connected called after pGWC-ZmCBL10-HA after definite obtained sequence that checks order is consistent with object fragment with pGWC-T carrier;

2) by pGWC-ZmCBL10-HA carrier and plant expression vector pEarleyGate100 by the connection of recombinating of LR enzyme, then transform DH5 α competent cell, obtain recombinant clone, after PCR detects, send order-checking by mono-clonal, the clone's called after pEarley Gate100-ZmCBL10-HA that checks order correct;

3) carrier of preparation is transformed to Agrobacterium GV3101 bacterial strain;

4) utilize Agrobacterium GV3101 bacterial strain conversion of plant plant, obtain the positive seedling strain of the conversion of isozygotying;

5) transgenic line is cultivated respectively on the MS substratum that contains 0mM, 125mM, 150mM NaCl, obtained the transfer-gen plant of Salt And Alkali Tolerance.

Beneficial effect of the present invention is:

The present invention finds that the albumen of ZmCBL10 coding is by regulation and control downstream genes involved involved in plant Salt And Alkali Tolerance process first.Further making ZmCBL10 gene of the present invention carry out involved in plant salt tolerant process by gene regulating becomes possibility, significant for the transgenic crop of cultivating high yield.

Brief description of the drawings

Fig. 1 is plant expression vector schematic diagram in the embodiment of the present invention 1.

Fig. 2 is that the PCR of ZmCBL10 transgenic line goal gene in the embodiment of the present invention 3 detects and western detects;

In Fig. 2 A, the different transgenic line goal gene of 1-10:ZmCBL10 amplification; WT: negative contrast (taking the DNA of Arabidopis thaliana wild-type Col as pcr template); +: positive control (taking ZmCBL10 overexpression vector as pcr template); Gene: be the expansion fragment of ZmCBL10; Bar: be the expansion fragment of Bar gene.

In Fig. 2 B, WT: negative contrast (taking the total protein of Arabidopis thaliana wild-type Col as loading object); The different transgenic lines of L1-L10:ZmCBL10 (taking the total protein of different transgenic lines as loading object); HA antibody: with the antibody of target protein specific combination; Actin antibody: for the protein bound antibody of Arabidopis thaliana Actin.

Fig. 3 is transgenic arabidopsis and the growing state comparison on the substratum that contains Different stress condition of wild-type plant in the embodiment of the present invention 4.

Fig. 4 is that the embodiment of the present invention 5 transfer ZmCBL10 gene Arabidopis thalianas can the responsive phenotype of complementary Arabidopis thaliana sos3 mutant under Different stress condition.

Turn the seed program request of complementary strain of ZmCBL10 gene on the MS of Different stress treatment condition substratum, 4 DEG C of vernalization are after 3 days, be placed in 22 DEG C of cultivations, within 10 days, observe afterwards the complementary strain of upgrowth situation and the growing state of mutant on different treatment condition flat board, complementary strain (COM-sos3) growing way is obviously better than sos3 mutant.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.

The structure of the expression vector that embodiment 1 contains ZmCBL10 gene

1,, according to the sequence (as shown in SEQ ID No.2) of known goal gene, design is with the primer of HA label tail end, from corn cDNA amplification ZmCBL10 gene order, and by sepharose test kit recovery object fragment.

2, the fragment reclaiming is connected with pGWC-T carrier, then transforms DH5 α competent cell, obtain positive monoclonal, send order-checking, through sequence alignment, obtain the mono-clonal consistent with former sequence, shake bacterium and extract this mono-clonal plasmid, by its called after pGWC-ZmCBL10-HA.

3, pGWC-ZmCBL10-HA and pEarleyGate100 plasmid are mixed to the rear LR of interpolation enzyme, after reacting by LR, obtain recombinant vectors pEarleyGate100-ZmCBL10-HA (as shown in Figure 1).Transform afterwards DH5 α competent cell, send order-checking by the positive monoclonal of acquisition, confirm errorless through sequence alignment.

The conversion that embodiment 2 contains CBL10 gene and screening

1, the ZmCBL10 overexpression vector building proceeds to Agrobacterium GV3101.

2, by the method arabidopsis thaliana transformation wild-type of being stained with flower dip-dye (floral dipping).

3, by the T1 that obtains after transforming for seed vernalization 3 days, be then directly seeded in Nutrition Soil and grow, normal growth is after approximately two weeks, PPT (phosphinothricin) the sprinkling T1 with 0.5 ‰ screens transformed plant for Arabidopis thaliana.

4, because plant overexpression vector used is with the Basta gene of anti-PPT (phosphinothricin), it can proceed to plant materials with goal gene in the time transforming, so spraying most of unconverted plant death after PPT (phosphinothricin), and transformed plant still can normal growth.Transformed plant is gathered in the crops respectively T2 for seed according to different strains.

5, gather in the crops each transgenic line T2 for seed after, the NaClO with 0.5% is to seed disinfection.Then dibbling (each transgenic line need to approximately 50 seeds) in the MS culture medium flat plate that contains 0.5 ‰ PPT (phosphinothricin), taking wild-type as negative contrast.

6, vernalization is placed in 22 DEG C of illumination boxs and grows for three days, observes growth of seedling situation after one week.Wild-type cannot be survived in the MS substratum of the PPT (phosphinothricin) containing 0.5 ‰; T2, therefore can occur by some seed without PPT (phosphinothricin) resistance owing to can separating and independent assortment by producer in the process that produces filial generation for heterozygote transgenic line; Only have T2 on behalf of the seed of homozygote transgenic line can be all surviving containing in the MS substratum of PPT (phosphinothricin) resistance.

The transfer-gen plant nucleic acid PCR analysis that embodiment 3 contains corn C BL10 gene

1, taking total DNA of the positive plant that extracts as template, carry out pcr amplification with ZmCBL10 gene primer, positive plant can amplify the band of 642bp, and wild-type plant can not amplify object band, as shown in Figure 2 A.Westernblot detected result as shown in Figure 2 B.

2, gather in the crops each transgenic line T2 for seed after, the NaClO with 0.5% is to seed disinfection.Then dibbling (each transgenic line need to approximately 50 seeds) in the MS culture medium flat plate that contains 0.5 ‰ PPT (phosphinothricin), taking wild-type as negative contrast.

3, vernalization is placed in 22 DEG C of illumination boxs and grows for three days, observes growth of seedling situation after one week.Wild-type cannot be survived in the MS substratum of the PPT (phosphinothricin) containing 0.5 ‰.

4, T2, for heterozygote transgenic line owing to can separating and independent assortment by producer in the process that produces filial generation, therefore can occur by some seed without PPT (phosphinothricin) resistance; Only have T2 on behalf of the seed of homozygote transgenic line can be all surviving containing in the MS substratum of PPT (phosphinothricin).

The transfer-gen plant that embodiment 4 contains CBL gene and the comparison of wild-type plant resistance

By the dibbling of ZmCBL10 transgenic line, on the MS of the NaCl that contains 0mM, 125mM, 150mM flat board, 4 DEG C of vernalization 3 days, is placed in 22 DEG C, in hour dark incubator in 16 little time/8, and photographic analysis (as shown in Figure 3) after 14 days.From result, can find out, along with the increase wild-type of NaCl concentration is being coerced lower cotyledon hypoevolutism and seedling greening-rate reduction, the growing way of transgenic line is obviously better than wild-type plant.In addition both in normal MS substratum phenotype without significant difference.

Responsive phenotype can recover it after embodiment 5 corn ZmCBL10 gene transformation Arabidopis thaliana sos3 Different stress is processed time

1, pEarleyGate100-ZmCBL10 overexpression vector is proceeded to Agrobacterium GV3101, with the method arabidopsis thaliana transformation sos3 mutant of being stained with flower dip-dye (floral dipping).

2, by the T1 that obtains after transforming for seed vernalization 3 days, be then directly seeded in Nutrition Soil and grow, normal growth is after approximately two weeks, PPT (phosphinothricin) the sprinkling T1 with 0.5 ‰ screens transformed plant for Arabidopis thaliana.The plant that can survive is T1 for plant.

3, gather in the crops respectively seed according to different strains.With PPT screening, statistics survival strain number, gets and meets the strain that 3:1 separates, and is the T2 of single copy for plant.

4, T2 is moved to small flower breeding for the plant of survival, results obtain T3 seed, continued to screen on the MS substratum that contains PPT, the homozygote strain that acquisition can be survived completely, gathering in the crops its seed is the complementary strain of transgenosis (COM-sos3).

5, will turn on the MS flat board of the NaCl that ZmCBL10 gene complementation strain and the program request of mutant strain contain 0mM, 75mM, 100mM and 125mM, 4 DEG C of vernalization 3 days, be placed in 22 DEG C, in the incubator of 16 little time/8 hour dark, after 10 days, observe phenotype and carry out photographic analysis (as shown in Figure 4).From result, can find out, mutant cotyledon hypoevolutism under Different stress is processed is even difficult to sprout, and complementary strain shows with wild-type suitable.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (8)

1. corn calcium adjusting phosphatase B analogy albumen ZmCBL10 gene, is characterized in that, the aminoacid sequence of its coding is as shown in SEQ ID No.1.

2. ZmCBL10 gene according to claim 1, is characterized in that, the nucleotide sequence of its encoding sequence is as shown in SEQ ID No.2.

3. the primer pair of ZmCBL10 gene coded sequence described in amplification claim 1 or 2, is characterized in that, described primer pair comprises:

Forward primer F:5'-atgccacgagccactgaggcggctt-3';

Reverse primer R:5'-ctaatcttccaccgccgtgttg-3'.

4. contain the carrier of gene described in claim 1 or 2.

5. contain the engineering bacteria of gene described in claim 1 or 2.

6. the application of the gene described in claim 1 or 2 in improvement plant anti-salt is coerced.

7. application according to claim 6, is characterized in that, gene described in claim 1 or 2 is transformed in plant plant by carrier or engineering bacteria, through screening, obtains the transgenic plant plant of improvement salt stress resistance.

8. application according to claim 7, is characterized in that, specifically comprises the steps:

1) taking corn cDNA as template, utilize primer pair amplification ZmCBL10 gene order described in claim 3, after adding HA label, sequence end is connected called after pGWC-ZmCBL10-H A after definite obtained sequence that checks order is consistent with object fragment with pGWC-T carrier;

2) by pGWC-ZmCBL10-HA carrier and plant expression vector pEarleyGate100 by the connection of recombinating of LR enzyme, then transform DH5 α competent cell, obtain recombinant clone, after PCR detects, send order-checking by mono-clonal, the clone's called after pEarleyG ate100-ZmCBL10-HA that checks order correct;

3) carrier of preparation is transformed to Agrobacterium GV3101 bacterial strain;

4) utilize Agrobacterium GV3101 bacterial strain conversion of plant plant, obtain the positive seedling strain of the conversion of isozygotying;

5) transgenic line is cultivated respectively on the MS substratum that increases successively salt concn, obtained the transfer-gen plant of Salt And Alkali Tolerance.