CN102827263A - Application of MuMADS2 to culturing of fruit quality-improved transgenic plant - Google Patents
Application of MuMADS2 to culturing of fruit quality-improved transgenic plant Download PDFInfo
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Abstract
The invention provides an application of MuMADS2 to culturing of a fruit quality-improved transgenic plant. The invention provides an application of a MuMADS2 protein or an encoding gene of the MuMADS2 protein to regulation and control of a plant phenotype, improvement on the plant fruit quality and/or increase in a gene expression quantity which is relevant to fruit maturity; and the amino acid sequence of the MuMADS2 protein is a sequence 2 in a sequence table. As proved by the results of reduction in the plant height and reduction in the quantity of fruit seeds in an experiment in which the MuMADS2 is introduced into a tomato for obtaining seven independent strains of a MuMADS2 transgenic tomato driven by a 35S promoter and the MuMADS2 is excessively expressed, the MuMADS2 plays an important role in the processes of fruit development and quality forming.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the application of a kind of MuMADS2 in the transgenic plant of cultivating fruit quality improving.
Background technology
The protein of MADS-box genes encoding is one type of huge transcription factor family of number very conservative on evolving; At first isolated MADS-box gene is that isolated DEFICENS (DEF) reaches isolated AGAMSUS (AG) gene from Arabidopis thaliana from Common Snapdragon in the plant; Subsequently through continuous research to these two kinds of model plants; Find that the MADS-box gene plays an important role in the growth course of flower, regulated and control plant from the growth of the formation of flowering time, floral organ, pollen and fertility or the like.
Ripening of fruits is a very complicated change procedure; Comprised the change of a series of physiological and biochemical procedures, like formation of secondary meta-bolitess such as cell wall structure variation, starch and glycometabolic change (changes in sugar/starch metabolism), pigment etc.These variations are results of multiple heredity and biochemical reactions in the plant materials.Fruit is divided into transition type fruit and non-transition type fruit according to the characteristics that ripening process ethene produces; Transition type fruit model plant such as tomato; Acquisition through the ripe two mutants rin/cnr/nor of tomato obtains deeply the research of fruit maturation regulatory gene; Confirmed that ethene playing the part of important role in ripening of fruits, this hormone is the pacing items that realizes tomato and other transition type fruit maturation.The sophisticated LeMADS-RIN of control is MADS box SEPALLATA (SEP) family member in the tomato, is that tamato fruit is ripe essential.Discoveries such as Vrebalov have caused the inactivation of two placed in-line MADS-box genes, i.e. LeMADS-RIN and LeMADS-MC owing to the DNA that the tomato dna group has lacked one section 3kb has obtained the rin two mutants.The sudden change of LeMADS-RIN makes the fruit can not be ripe, and the expression analysis result on the transcriptional level shows that LeMADS-RIN mainly expresses in fruit, and expresses enhancing with fruit maturation.The same year; People such as Vrebalov have isolated homologous gene Fv-MADS-9 again from non-transition type fruit strawberry; This gene presents the single-minded expression pattern of fruit, and proves that the MADS-box gene is the upper reaches regulatory factor of fruit maturation, regulates and control the maturation of transition type fruit and non-transition type fruit simultaneously.Evidence shows that RIN albumen can infer that thus RIN MADS-box transcription factor possibly be to come the sophisticated of regulating fruit through the expression of regulating and control ACS2 with the rare biosynthesizing key gene of the second of tomato---the cis-acting elements of ACS2 combines in the body.6 MADS-box gene M aMADS1-MaMADS6 have been cloned in (2010) such as Elitzur T from banana; Studied them respectively and grown at banana and adopt after ripening process mesocarp and pulp and reach expression under ethene is induced etc., the result shows that what take place in pericarp and the pulp is two relatively independent ripening processs.In pulp, mainly be and MaMADS4, relevant with 5 and the activation of the MaMADS1 in later stage, and in pericarp, mainly be relevant with the transcriptional activation of MaMADS1 and 3.Therefore, this type transcription factor regulated and control network that possibly participate in ripening process is being controlled the maturation of fruit.
Banana (Musa cuminata) is important tropical fruit, and fruit is typical ethene transition type fruit, along with the formation fruit of adopting back ethene is accomplished the after-ripening process and ripe rapidly aging.
Summary of the invention
An object of the present invention is to provide the new purposes of MuMADS2 albumen or the proteic encoding sox of MuMADS2.
The invention provides the application of the proteic encoding sox of MuMADS2 albumen or MuMADS2 in regulation and control plant phenotype, improvement fruit quality and/or raising and fruit maturation related gene expression amount;
The proteic aminoacid sequence of said MuMADS2 is the sequence 2 in the sequence table.
In the above-mentioned application, said regulation and control plant phenotype is embodied in and reduces the plant plant height, reduces fruit weight and/or reduce plant seed quantity;
Said improvement fruit quality is embodied in amino acid, Serlabo, Lyeopene, xenthophylls and/or the ascorbic acid content that improves fruit;
Said amino acid is specially aspartic acid and/or L-glutamic acid;
Said and fruit maturation genes involved are at least a among ACS2, CNR, E8, PG, PSY1 and the TAGL1;
Said plant is specially dicotyledons or monocotyledons; Said dicotyledons further is specially tomato;
The proteic encoding sox of said MuMADS2 is that sequence 1 or the sequence 1 in the sequence table in the sequence table is from 5 ' terminal 1-702 position Nucleotide.
Another object of the present invention provides a kind of cultivation transgenic plant method.
Method provided by the invention for the proteic encoding sox of MuMADS2 is imported the purpose plant, obtains to have following 1)-5) in the transgenic plant of at least a characteristic:
1) plant height of said transgenic plant is lower than said purpose plant;
2) fruit weight of said transgenic plant is less than said purpose plant;
3) seed amount of said transgenic plant is less than said purpose plant;
4) quality of said transgenic plant fruit obtains improvement;
5) the said and fruit maturation related gene expression amount in the said transgenic plant fruit is higher than said purpose plant;
The proteic aminoacid sequence of said MuMADS2 is the sequence 2 in the sequence table.
In the aforesaid method, the nucleotides sequence of the proteic encoding sox of said MuMADS2 is classified sequence 1 or the sequence 1 in the sequence table in the sequence table as from 5 ' terminal 1-702 position Nucleotide.
In the aforesaid method, the quality of said transgenic plant fruit obtains the now following A-E of modification:
The aminoacids content of A, said transgenic plant fruit is higher than said purpose plant;
The carotene carotene content of B, said transgenic plant fruit is higher than said purpose plant;
The content of lycopene of C, said transgenic plant fruit is higher than said purpose plant;
The lutein content of D, said transgenic plant fruit is higher than said purpose plant;
The ascorbic acid content of E, said transgenic plant fruit is higher than said purpose plant.
In the aforesaid method, said amino acid is aspartic acid and/or L-glutamic acid.
In the aforesaid method, the proteic encoding sox of said MuMADS2 imports said purpose plant through expression vector.
In the aforesaid method, said expression vector is the proteic encoding sox of said MuMADS2 to be inserted among the pCAMBIA1302 the proteic carrier of expression MuMADS2 that obtains; In an embodiment of the present invention, expression vector is specially the sequence in the sequence table 1 is inserted the carrier that obtains between NcoI and the SpeI restriction enzyme site of pCAMBIA1302 from 5 ' terminal 1-702 position Nucleotide.
In the aforesaid method, said purpose plant is dicotyledons or monocotyledons; Said dicotyledons is specially tomato.
The 3rd purpose of the present invention provides a kind of expression vector.
Expression vector provided by the invention is for inserting the proteic encoding sox of MuMADS2 among the pCAMBIA1302 the proteic carrier of expression MuMADS2 that obtains; In an embodiment of the present invention, expression vector is specially the sequence in the sequence table 1 is inserted the carrier that obtains between NcoI and the SpeI restriction enzyme site of pCAMBIA1302 from 5 ' terminal 1-702 position Nucleotide.
The proteic aminoacid sequence of said MuMADS2 is the sequence 2 in the sequence table; The proteic encoding sox of said MuMADS2 is specially sequence 1 or the sequence 1 in the sequence table in the sequence table from 5 ' terminal 1-702 position Nucleotide.
Experiment of the present invention proves; The present invention imports MuMADS2 in the tomato; Obtain the commentaries on classics MuMADS2 tomato strain system that 35S promoter drives; Overexpression MuMADS2 has reduced plant height, has reduced fruit and seed quantity, crosses expression MuMADS2 and makes tamato fruit ACS2, E8, TAGL1, PG, CNR, the isogenic expression of PSY1 significantly strengthen; Compare with the wild-type tomatoes fruit, the essential amino acids content of transgenic Fructus Lycopersici esculenti fruit changes not obvious, but aspartic acid and glutamic increase the content increase of fruit Serlabo and xitix, Lyeopene and xenthophylls; Sugar content is compared no noticeable change with wild-type.These results show that MuMADS2 is bringing into play important effect in fruit development and quality forming process.
Description of drawings
Fig. 1 is that MuMADS2 is at the RT-PCR of banana different tissues expression analysis
Fig. 2 detects for the Southern trace
Fig. 3 is for changeing MuMADS2 tomato morphological observation
Fig. 4 is the regulation and control of MuMADS2 to fruit shape, size
Fig. 5 is the regulation and control of MuMADS2 to fertility
Fig. 6 is that MuMADS2 is in fruit development different times expression of results
Fig. 7 is for changeing the ripe related gene expression of MuMADS2 tamato fruit
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Banana among the following embodiment (Musa acuminate L.AAA group cv.Brazilian) fruit (blooming back 100-120 days) is bought from torrid zone biotechnology research institute of Chinese Academy of Tropical Agricultural Sciences Chengmai banana plantation.It is subsequent use that the fruit that picks up from green ripe stage (spending back 100 days) is adopted in back fruit, male flower, female flower, petal, stamen, style and column cap, ovary, root, stem and the Ye Yu liquid nitrogen in different ripening stages after the quick-frozen-80 ℃ of preservations.
Tomato (Lycopersivonesculentum Mill.) is designated hereinafter simply as wild-type tomatoes, available from quality resource institute of Chinese Academy of Tropical Agricultural Sciences vegetables center for the AV6 pure lines).Be planted in China tropic Agriculture Academy Sciences tropic Biotechnology Research Institute experiment base ,-80 ℃ of preservations are subsequent use behind fruit development different times (underdone green fruit, green ripe fruit, annesl fruit, red ripe fruit) collecting fruit and the blade liquid nitrogen flash freezer.
The acquisition of embodiment 1, commentaries on classics MuMADS2 tomato
1, the acquisition of gene M uMADS2
The structure banana is adopted the early stage reduction library (SSH) that suppresses, back, obtains one and MADS-box family gene homologous cDNA fragment.According to this cDNA fragment sequence design 5 ' and 3 ' end primer; Utilize RACE technology amplification 5 ' and 3 ' end, splicing obtains 1 banana MADS-box gene, according to splicing consequence devised gene 5 ' and 3 ' end primer; With the total RNA reverse transcription of banana cDNA as template; Amplification, the nucleotides sequence of the PCR product that obtains is classified the sequence 1 in the sequence table as, and the unnamed gene shown in this nucleotide sequence is MuMADS2; Its encoded protein is MuMADS2, and this proteic aminoacid sequence is the sequence 2 in the sequence table.
2, banana MuMADS2 expression analysis
Adopt back banana (choosing the fruit of adopting back 7 mature periods) and other organ and carry out total RNA extraction, cDNA reverse transcription.
At non-conserved regions design 5 ' end primer 5 '-GTCCTCTAAGTGTCAAGGAGC-3 ', 3 ' end primer 5 '-CTGATTCTGCTGCATGAAGTT-3 ' carries out RT-PCR, real-time qRT-PCR analyzes according to the MuMADS2 sequence.With ACTIN is confidential reference items, and the primer sequence of amplification confidential reference items is A15 ': 5 '-AGGCAGGATTTGCTGGTGA-3 ' and A23 ': 5 '-ACCAGTGGTACGACCGCTA-3 ', and amplification length is 398bp.
The result is as shown in Figure 1, and MuMADS2 is at the RT-PCR of banana different tissues expression analysis, wherein, and M: male flower; F: female flower; Pe: petal; St: stamen; Sty/sti: style and column cap; Ov: ovary; F: fruit; R: root; S: stem; L: leaf; Can find out that this gene is mainly expressed in the ovary of female flower and male flower and fruit, in other tissue, do not express or expression amount seldom.
3, the amplification of expression vector
Extract the banana leaf DNA, with M2GFP5 ': CG
GAAGGGGTAAG; M2GFP3 ': GCG
TGCAGCAGAATCAGT is a primer, obtains the PCR product of about 702bp.
The PCR product that obtains is cut with NcoI and SpeI enzyme; The enzyme that obtains is cut product and the pCAMBIA1302 carrier (Subcellular Localization of banana MuMADS2 gene expression product of cutting through same enzyme; 2010, tropical crops journal, the 31st the 9th phase of volume; 777-781, the public can obtain from Chinese Academy of Tropical Agricultural Sciences's torrid zone biotechnology research.) connect, obtain connecting product, will connect the product transformed into escherichia coli, obtain transformant.Extract the plasmid of transformant, send to order-checking, the result is the carrier of this plasmid for the sequence in the sequence table 1 is obtained, called after pCAMBIA1302-MuMADS2 between the NcoI of 5 ' terminal 1-702 position Nucleotide insertion pCAMBIA1302 and SpeI restriction enzyme site.
4, the acquisition of reorganization bacterium
Change pCAMBIA1302-MuMADS2 over to the Agrobacterium tumefaciens LBA4404 (Subcellular Localization of banana MuMADS2 gene expression product; 2010; The tropical crops journal; The 31st the 9th phase of volume, 777-781, the public can obtain from Chinese Academy of Tropical Agricultural Sciences's torrid zone biotechnology research.) in, obtain the bacterium of recombinating, extract the plasmid order-checking of reorganization bacterium, this plasmid is pCAMBIA1302-MuMADS2, will contain the reorganization bacterium called after LBA4404/pCAMBIA1302-MuMADS2 of this plasmid.
5, change acquisition and the evaluation of MuMADS2 tomato
1) changeing the MuMADS2 tomato obtains
Above-mentioned LBA4404/pCAMBIA1302-MuMADS2 is infected the blade of wild-type tomatoes, and Agrobacterium has been infected about 700 leaf dishes.The bud that differentiates obtains 45 resistant budses through screening, last transplant survival 37 strain resistant plants, transformation efficiency is 5.29%; Obtain 37 strain T0 for changeing the MuMADS2 tomato.
2) evaluation of commentaries on classics MuMADS2 tomato
Extracting T0 and detect for the Southern trace that carries out that changes the MuMADS2 tomato, is contrast with wild-type tomatoes (WT), and probe primer is M2probe1:5 '-TCAGGTGCCATTGTAGAT-3 ' and M2probe2:5 '-GTCATAGCCAAGATGGAG-3 '.The result is as shown in Figure 2; 27,15,53,26,28,13,56,57,23,12,4,8 is that T0 is for the not homophyletic system that changes the MuMADS2 tomato; WT is a wild-type tomatoes; Can find out, obtain positive T0 that 9 strains are numbered L53, L56, L57, L15, L27, L4, L13, L12, L26 for changeing the MuMADS2 tomato.
Adopting uses the same method changes empty carrier pCAMBIA1302 in the wild-type tomatoes over to, obtains T0 for changeing the empty carrier tomato, carries out the Southern trace according to the method described above and detects, and does not detect the purpose fragment, explains to obtain positive T0 for changeing the empty carrier tomato.
Above-mentioned T0 is all sowed, receives kind for plant, up to obtaining T2 for homozygous lines.
The functional study of embodiment 2, commentaries on classics MuMADS2 tomato
1, changes the phenotype of MuMADS2 tomato
Sowing is numbered the T2 of L12, L13, L15, L26, L27, L53, L56 for changeing MuMADS2 tomato (MuMADS2), T2 for changeing empty carrier tomato and wild-type tomatoes (contrast); With above strain is that tomato seeds is broadcast in pot for growing seedlings; At 25 ℃, illumination 3000lux, 8h/d cultivates down; Grow about 2 months when the plant strain growth stalwartness, change seedling over to land for growing field crops when plant height reaches 15-20cm.Each strain is 20 strains, experiment triplicate, results averaged ± standard deviation.
Result such as Fig. 3-shown in Figure 5, wherein Fig. 3 is a morphological observation, and Fig. 4 is the regulation and control of MuMADS2 to fruit shape, size, and Fig. 5 is the regulation and control of MuMADS2 to fertility;
Find out among Fig. 3 that T2 compares the dwarfism that shows in various degree for changeing the MuMADS2 tomato plant with wild-type tomatoes; Aspect flower development, indivedual strains are that the flower pesticide variation develops into petal or gynoecium, and ovary obviously increases; Transgenic Fructus Lycopersici esculenti L53 shows dwarfing, and stem is sturdy and upright, and growth is compact; The internode cripetura; The bud of growing thickly; The blade outward appearance is also quite special, and leaf margin is level and smooth and curling to leaf reverse side direction, and mostly the blade of contrast strain is decomposite leaf, and blade quantity and size also all are higher than transfer-gen plant, light green; Fruit oval, seed amount are starkly lower than the contrast strain, or even sterile;
The statistics plant height; The result is following: the height that is numbered 7 transgenic lines of L12, L13, L15, L26, L27, L53, L56 be respectively (unit: cm): 113.25 ± 4.03,54.25 ± 11.44,57.25 ± 4.65,78.75 ± 13.15,55.25 ± 2.50,44.75 ± 6.95,48.25 ± 7.23, the plant height of wild-type tomatoes is 126.75 ± 3.50cm;
Find out among Fig. 4 that T2 compares the fruit weight saving that diminishes for changeing the MuMADS2 tomato plant with wild-type tomatoes; Statistics fruit heavy (fruit weight); The result is following: 7 T2 that are numbered L12, L13, L15, L26, L27, L53, L56 are respectively (unit: restrain) for the single fruit weight that changes the MuMADS2 tomato: 4.51 ± 1.47,2.72 ± 0.62,3.69 ± 0.61,5.13 ± 0.77,5.59 ± 0.42,6.24 ± 0.97,5.07 ± 0.81, and the wild-type tomatoes fruit weighs 6.33 ± 1.03 grams;
Among Fig. 5, A is the green ripe stage wild-type tomatoes, and B is that green ripe stage T2 is for changeing the MuMADS2 tomato; C is red ripe phase wild-type tomatoes; D is red ripe phase T2 for changeing the MuMADS2 tomato, can find out, T2 compares seed amount and reduces for changeing the MuMADS2 tomato plant with wild-type tomatoes.
The seed amount of adding up the red ripe phase is following: the seed amount that is numbered 7 transgenic lines of L12, L13, L15, L26, L27, L53, L56 all reduces; The seed grain number that 7 strains are fruit is respectively: 22.47 ± 9.23,7.87 ± 3.89,22.47 ± 9.23,23.60 ± 6.94,2.00 ± 0.02,0 ± 0,5.2 ± 2.60, and wild-type tomatoes is 48.60 ± 8.92 of each fruits;
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
2, tamato fruit correlation of attributes index determining
Gather the T2 that is numbered L56, L13, L26 for changeing MuMADS2 tomato, T2 for (red mature-RM) fruit of red ripe phase that changes empty carrier tomato and wild-type tomatoes, measure aminoacids content, sugared content, Serlabo, Lyeopene, xitix and lutein content these and correlation of attributes index respectively.Each strain is 3 strains, like no specified otherwise, and experiment triplicate, results averaged.
The detection method of aminoacids content is documented in Yu Shan et al.2010 AMINO-ACID AND MINERAL COMPOSITION OF Stellaria media.Chemistry of Natural Compounds, Vol.46, No.4;
The sugar content detecting method is documented in Mateja C et al.2006Influence of branch bending on sugar organic acid and phenolic content in fruits of ' Willians ' pear (Pyruscommunis L.) .J Sci Food Agric 86:2463-2467;
The detection method of Serlabo is documented in Olives Barba et al, 2006 Application of a UV – vis detection-HPLC method for a rapid determination of lycopene and b-carotene in vegetables.Food Chemistry, 95328 – 336;
The detection method of Lyeopene is documented in Olives Barba et al, 2006 Application of a UV – vis detection-HPLC method for a rapid determination of lycopene and b-carotene in vegetabl es.Food Chemi stry, 95328 – 336;
The detection method of xitix is documented in Michael Tausz; Et al.1996; Simultaneous Determination of Ascorbic Acid and Dehydroascorbic Acid in Plant Materials by High Performance Liquid Chromatography.Phytochemical analysis, vol.7.69-721996;
The detection method of xenthophylls is documented in Brendon D.Gill et al.2008, Liquid chromatographic method for the determination of lutein in milk and pediatric formulas.Internat ional Dairy Journal 18894 – 898;
1) 17 seed amino acid content
Detection is numbered the T2 of L56 for changeing MuMADS2 tomato and (red mature-RM) fruit result of red ripe phase of wild-type tomatoes shown in table 1-table 2:
Table 1 is 17 seed amino acid content of wild-type tomatoes
Table 2 is to be numbered the T2 of L56 for the 17 seed amino acid content that change the MuMADS2 tomato
Can find out that from table 1 and table 2 T2 that is numbered L56 is higher than wild-type tomatoes for the aspartic acid (Asp) among the commentaries on classics MuMADS2 and the content of L-glutamic acid (Glu), other aminoacids content changes not remarkable.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
2) sugar degree
The T2 that is numbered L56 is following for the red ripe phase fruit glucose and the fructose content that change MuMADS2 tomato (L56) and wild-type tomatoes (CK):
Wild-type tomatoes fruit glucose and fructose content are respectively 37.4472 and 108.9328,
The T2 that is numbered L56 is respectively 37.0648 and 108.288 for changeing MuMADS2 tamato fruit glucose and fructose content.
Can find out that glucose and fructose content in numbering L56 and the wild-type tomatoes fruit do not have remarkable difference.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
3) carotene carotene content
The T2 that is numbered L56, L13, L26 is following for carotene carotene content result in commentaries on classics MuMADS2 tomato and the wild-type tomatoes fruit:
The T2 that is numbered L56 is for the carotene carotene content 5.1711mg/kg that changes in the MuMADS2 tomato;
The T2 that is numbered L13 is for the carotene carotene content 4.48mg/kg that changes in the MuMADS2 tomato;
The T2 that is numbered L26 is for the carotene carotene content 9.27mg/kg that changes in the MuMADS2 tomato;
Carotene carotene content 4.0348mg/kg in the wild-type tomatoes;
Can find out, compare that T2 increases for the Serlabo that changes MuMADS2 tomato transgenic plant with wild-type tomatoes.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
4) content of lycopene
The T2 that is numbered L56, L13, L26 is following for the content of lycopene result who changes in MuMADS2 tomato and the wild-type tomatoes fruit:
The T2 that is numbered L56 is for the content of lycopene 64.8278mg/kg that changes in the MuMADS2 tomato;
The T2 that is numbered L13 is for the content of lycopene 56.37mg/kg that changes in the MuMADS2 tomato;
The T2 that is numbered L26 is for the content of lycopene 52.62mg/kg that changes in the MuMADS2 tomato;
Content of lycopene 43.0320mg/kg in the wild-type tomatoes;
Can find out, compare that T2 significantly increases for the content of lycopene that changes MuMADS2 tomato transgenic plant with wild-type tomatoes.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
5) lutein content
The T2 that is numbered L56, L13, L26 is following for the lutein content result who changes in MuMADS2 tomato and the wild-type tomatoes fruit:
Lutein content in wild-type tomatoes (CK) fruit is 0.6014mg/kg,
The T2 that is numbered L56 is 1.7388mg/kg for the lutein content that changes in the MuMADS2 tamato fruit,
The T2 of numbering L13 is 1.001mg/kg for the lutein content that changes in the MuMADS2 tamato fruit;
The T2 of numbering L26 is 1.29mg/kg for the lutein content that changes in the MuMADS2 tamato fruit;
Can find out, compare that T2 significantly increases for the lutein content that changes MuMADS2 tomato transgenic plant with wild-type tomatoes.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
6) ascorbic acid content
The T2 that is numbered L56, L13, L26 is following for the ascorbic acid content result who changes in MuMADS2 tomato and the wild-type tomatoes fruit:
Ascorbic acid content in wild-type tomatoes (CK) fruit is 20.0934mg/kg,
The T2 that is numbered L56 is 28.0261mg/kg for the ascorbic acid content that changes in the MuMADS2 tamato fruit,
The T2 of numbering L13 is 26.31mg/kg for the ascorbic acid content that changes in the MuMADS2 tamato fruit;
The T2 of numbering L26 is 22.40mg/kg for the ascorbic acid content that changes in the MuMADS2 tamato fruit;
Can find out, compare that T2 increases for changeing MuMADS2 tomato transgenic plant ascorbic acid content with wild-type tomatoes.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
3, MuMADS2 expresses at the fruit development different times
Gather respectively be numbered L12, L13, L15, L26, L27, L53, L56 T2 for changeing the MuMADS2 tomato at young fruit period immature green (IGM), green ripe stage green mature (GM), Veraison breaker fruit (BR) and 4 growth period fruits of red ripe phase red mature (RM); Extract total RNA; Reverse transcription cDNA utilizes forward primer; 5 '-GTCCTCTAAGTGTCAAGGAGC-3 ' and reverse primer, 5 '-CTGATTCTGCTGCATGAAGTT-3 ' carry out the expression that qRT-PCR detects MuMADS2.The internal control gene accession number is (AK318637), and primer sequence is actinFW:5'-CCAACAGAGAGAAGATGA-3'actinRV:5'-ATGTCTCTTACAA TTTCCCG-3'.
The result is as shown in Figure 6, wherein detects less than this expression of gene basically in L15 and the L27 fruit, and L53 and L57 strain are that expression amount is very low in the fruit, and other strain system all detects the expression of goal gene, and wherein L56, L13, L12 and L26 expression amount are higher.Different transgenic lines differ greatly in the different times expression amount variation of fruit development, and L56 and L13 are big at GM and BR expression amount in period, and L12 and L26 are only big at the GM expression amount.This possibly be because not due to the site difference of the copy number that inserts of homophyletic system and insertion.Because homophyletic is not that genetic expression and fruit development show different characteristic, infer that they form the influence difference to fruit development and quality.
4, the ripe related gene expression of tamato fruit
With fruit maturation closely-related ethylene metabolism pathway gene ACS2 and E8; Hardness of fruit development related gene PG; Correlation of attributes gene C NR and PSY1; And the important transcription factor TAGL1 that influences fruit maturation in the tomato.The T2 that detected wild-type tomatoes, is numbered L56 is 4 above-mentioned Expression of Related Genes of developmental stage of fruit for changeing the MuMADS2 tomato strain.
According to document (Julia Vrebalov; Irvin L.Pan; Antonio JavierMatas Arroyo; Et al2009 Fleshy Fruit Expansion and Ripening Are Regulated by the Tomato SHATTERPROOF Gene TAGL1.The Plant Cell, 21:3041 – 3062; ) in the ripe genes involved ACS2 of the tamato fruit that provides, CNR, E8, PG, PSY1 and TAGL1 gene primer sequence synthesized primer thing; Extraction is numbered L56T2 for changeing MuMADS2 tomato young fruit period immature green (IGM), fruit green ripe stage green mature (GM), fruit Veraison breaker fruit (BR) and 4 total RNA reverse transcriptions of growth period fruit of the red ripe phase red mature of fruit (RM) cDNA, and fluorescence quantitative RT-RCR (real-time qRT-PCR) is analyzed above-mentioned expression of gene characteristic.The internal control gene accession number is (AK318637), and primer sequence is actinFW:5 '-CCAACAGAGAGAAGATGA-3'actinRV:5 '-ATGTCTCTTACAATTTCCCG-3'.Is contrast with wild-type tomatoes and T2 for changeing the empty carrier tomato.
The result is as shown in Figure 7, and A is for being numbered L56T2 for changeing the MuMADS2 tomato, and B is a wild-type tomatoes, and concrete outcome is following:
ACS2 is lower at wild-type tamato fruit expression amount, and high expression level amount is at fruit Veraison (breaker fruit) 3.2; Reach 5 for changeing MuMADS2 tamato fruit Veraison (breaker fruit) expression amount being numbered L56T2.
CNR is 2.3 in the identical high expression level amount of wild-type tomatoes at fruit Veraison (breaker fruit) expression amount; To reach peak at fruit green ripe stage (green mature) be 10.49 for changeing the MuMADS2 tomato being numbered L56T2, is 6.27 at fruit Veraison (breaker fruit) expression amount, also big than wild-type expression amount.
E8 is lower at wild-type tamato fruit expression amount, and high expression level amount is 2.7 at fruit Veraison (breaker fruit); Is 2.9 being numbered L56T2 for changeing MuMADS2 tamato fruit Veraison (breaker fruit) expression amount.
At fruit Veraison (breaker fruit), expression amount is 2 to PG in the identical high expression level amount of wild-type tomatoes; Is 43 being numbered L56T2 for changeing MuMADS2 tamato fruit Veraison (breaker fruit) relative expression quantity.
PSY1 is 1.7 at wild-type tamato fruit green ripe stage (green mature); Is 310 being numbered L56T2 for the fruit green ripe stage (green mature) that changes the MuMADS2 tomato.
At fruit Veraison (breaker fruit), expression amount is 15 to TAGL1 in the identical high expression level amount of wild-type tomatoes; Is fruit green ripe stage (green mature) being numbered L56T2 for changeing MuMADS2 tomato peak expression, and expression amount is 50, is 40.1 at fruit Veraison (breaker fruit) expression amount, and is also big than wild-type expression amount.
T2 does not have significant difference for changeing empty carrier tomato and wild-type tomatoes result.
Can find out that from above-mentioned MuMADS2 also shows significant regulating and controlling effect in the regulation and control sophisticated while of tamato fruit to other qualities of tomato.Anthocyan material in the transgenic Fructus Lycopersici esculenti fruit (Lyeopene, Serlabo) content increases.Secondly, xitix, aspartic acid (Asp) and L-glutamic acid (Glu) content all significantly increase in the fruit, explain that the MuMADS2 gene has regulating and controlling effect widely, are regulating numerous expression of target gene, influence numerous physiology of fruit and biochemical reaction.In a word, the result of study of this research discloses the MuMADS2 gene in the potential value to the fruit quality influence, might valuable genetic resources be provided for improveing fruit quality from now on, and the research to fruit quality formation mechanism simultaneously provides new point of penetration.
Claims (10)
1.MuMADS2 the application of the proteic encoding sox of albumen or MuMADS2 in regulation and control plant phenotype, improvement fruit quality and/or raising and fruit maturation related gene expression amount;
The proteic aminoacid sequence of said MuMADS2 is the sequence 2 in the sequence table.
2. application according to claim 1 is characterized in that:
Said regulation and control plant phenotype is embodied in and reduces the plant plant height, reduces fruit weight and/or reduce plant seed quantity;
Said improvement fruit quality is embodied in amino acid, Serlabo, Lyeopene, xenthophylls and/or the ascorbic acid content that improves fruit;
Said amino acid is specially aspartic acid and/or L-glutamic acid;
Said and fruit maturation genes involved are at least a among ACS2, CNR, E8, PG, PSY1 and the TAGL1;
Said plant is specially dicotyledons or monocotyledons; Said dicotyledons further is specially tomato;
The proteic encoding sox of said MuMADS2 is that sequence 1 or the sequence 1 in the sequence table in the sequence table is from 5 ' terminal 1-702 position Nucleotide.
3. cultivate the transgenic plant method for one kind,, obtain to have following 1 for the proteic encoding sox of MuMADS2 is imported the purpose plant)-5) in the transgenic plant of at least a characteristic:
1) plant height of said transgenic plant is lower than said purpose plant;
2) fruit weight of said transgenic plant is less than said purpose plant;
3) seed amount of said transgenic plant is less than said purpose plant;
4) quality of said transgenic plant fruit obtains improvement;
5) be higher than said purpose plant with fruit maturation related gene expression amount in the said transgenic plant fruit;
The proteic aminoacid sequence of said MuMADS2 is the sequence 2 in the sequence table.
4. method according to claim 3 is characterized in that: the nucleotides sequence of the proteic encoding sox of said MuMADS2 is classified sequence 1 or the sequence 1 in the sequence table in the sequence table as from 5 ' terminal 1-702 position Nucleotide.
5. according to claim 3 or 4 described methods, it is characterized in that:
The quality of said transgenic plant fruit obtains the now following A-E of modification:
The aminoacids content of A, said transgenic plant fruit is higher than said purpose plant;
The carotene carotene content of B, said transgenic plant fruit is higher than said purpose plant;
The content of lycopene of C, said transgenic plant fruit is higher than said purpose plant;
The lutein content of D, said transgenic plant fruit is higher than said purpose plant;
The ascorbic acid content of E, said transgenic plant fruit is higher than said purpose plant.
6. method according to claim 5 is characterized in that:
Said amino acid is aspartic acid and/or L-glutamic acid.
7. according to the arbitrary described method of claim 3-6, it is characterized in that:
The proteic encoding sox of said MuMADS2 imports said purpose plant through expression vector.
8. method according to claim 7 is characterized in that: said expression vector is for inserting the proteic encoding sox of said MuMADS2 among the pCAMBIA1302 the proteic carrier of expression MuMADS2 that obtains.
9. according to the arbitrary described method of claim 3-8, it is characterized in that: said purpose plant is dicotyledons or monocotyledons; Said dicotyledons is specially tomato.
10. expression vector is for inserting the proteic encoding sox of MuMADS2 among the pCAMBIA1302 the proteic carrier of expression MuMADS2 that obtains; The proteic aminoacid sequence of said MuMADS2 is the sequence 2 in the sequence table; The proteic encoding sox of said MuMADS2 is specially sequence 1 or the sequence 1 in the sequence table in the sequence table from 5 ' terminal 1-702 position Nucleotide.
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| CN108409858A (en) * | 2018-05-29 | 2018-08-17 | 天津农学院 | A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof |
| CN111676239A (en) * | 2020-06-23 | 2020-09-18 | 中国农业科学院郑州果树研究所 | Application of sweet cherry PaMADSs gene in regulation and control of fruit coloring or fruit ripening and softening |
| CN113907077A (en) * | 2021-09-30 | 2022-01-11 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | PSY1 mature polypeptide plant senescence inhibitor, preparation method and application thereof |
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| CN104611345B (en) * | 2015-02-05 | 2017-08-01 | 中国热带农业科学院热带生物技术研究所 | A kind of gene for improveing fruit quality and its coded product and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108409858A (en) * | 2018-05-29 | 2018-08-17 | 天津农学院 | A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof |
| CN108409858B (en) * | 2018-05-29 | 2021-06-18 | 天津农学院 | Tomato fruit transcription factor CNR polyclonal antibody and preparation method thereof |
| CN111676239A (en) * | 2020-06-23 | 2020-09-18 | 中国农业科学院郑州果树研究所 | Application of sweet cherry PaMADSs gene in regulation and control of fruit coloring or fruit ripening and softening |
| CN111676239B (en) * | 2020-06-23 | 2022-07-12 | 中国农业科学院郑州果树研究所 | Application of sweet cherry PaMADSs gene in regulation and control of fruit coloring or fruit ripening and softening |
| CN113907077A (en) * | 2021-09-30 | 2022-01-11 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | PSY1 mature polypeptide plant senescence inhibitor, preparation method and application thereof |
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| CN102827263B (en) | 2014-12-03 |
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