CN108384794A - Arabidopsis FtsHi5 genes are in the regulation and control developmental application of arabidopsis chloroplaset - Google Patents

Abstract

The invention discloses an application of the Arabidopsis thaliana FtsHi5 gene in regulating the development of the Arabidopsis thaliana chloroplast, and relates to the technical field of genetic engineering. The invention constructs the interference vector of Arabidopsis thaliana FtsHi5 gene, and then transfects wild type Arabidopsis thaliana to obtain the FtsHi5 interference mutant, which shows yellowish dwarf, decreased chlorophyll content, and damaged thylakoid structure in chloroplast when DEX is added. Experiments have proved that the Arabidopsis FtsHi5 gene is involved in the regulation of chloroplast development and can be used to regulate chloroplast development.

Description

Application of Arabidopsis thaliana FtsHi5 Gene in Regulation of Arabidopsis Chloroplast Development

technical field

The invention relates to the technical field of genetic engineering, in particular to the application of an Arabidopsis thaliana FtsHi5 gene in regulating the development of Arabidopsis chloroplasts.

Background technique

Chloroplasts are semi-autonomous organelles commonly found in terrestrial plants, algae, and some protists, and are the site of photosynthesis. Plant photosynthesis is the material basis for the survival and development of almost all organisms. Therefore, the study of chloroplast development and regulation mechanism will help to better understand the regulation mechanism of photosynthesis. Chloroplast is an important organelle in plants. Although its development and regulation mechanism have been widely concerned by researchers, due to its complexity, there are still many regulatory molecular mechanisms that are still unclear.

FtsH (Filamentation Temperature-Sensitive H) is a type of ATP-dependent metalloprotease widely present in prokaryotes and eukaryotes. There is also FtsHi (i: proteolyticinactivation) gene similar to FtsH in Arabidopsis, because it lacks Zn ²⁺ binding domain without protease activity. At present, there are few research reports on the biological function of FtsHi. FtsHi seems to be closely related to FtsH12, so it is speculated that its function is similar to FtsH12. In recent research reports, it was found that AtFtsHi1 is necessary for chloroplast development and embryogenesis. AtFtsHi1 may play a role in the process of plastid division and development, and is an essential gene for Arabidopsis chloroplast development. In addition, knockout of FtsHi2, FtsHi4, and FtsHi5 resulted in defects in embryonic development, similar to the phenomenon of embryonic lethality caused by knockout of FtsH12.

Contents of the invention

In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of Arabidopsis thaliana FtsHi5 gene in regulating the development of Arabidopsis chloroplasts.

The object of the present invention is achieved through the following technical solutions:

The invention provides an application of the Arabidopsis thaliana FtsHi5 gene in regulating the development of the Arabidopsis chloroplast.

Further, the present invention provides an application of Arabidopsis thaliana FtsHi5 gene in plant breeding.

Further, the present invention provides an application of Arabidopsis thaliana FtsHi5 gene in breeding transgenic plants.

Said plants include, but are not limited to, Arabidopsis, rice, wheat, corn, sorghum, millet, sugar cane, cotton, tomato, alfalfa and elephant grass;

The amino acid sequence of the Arabidopsis FtsHi5 gene is shown in SEQ ID NO:2.

The sequence of the Arabidopsis FtsHi5 gene is one of the following nucleotide sequences:

1) The DNA sequence shown in SEQ ID NO: 1 in the sequence listing;

2) the DNA sequence of the protein encoded by SEQ ID NO: 1 in the sequence listing;

Compared with the prior art, the present invention has the following advantages and effects:

The invention constructs the interference vector of Arabidopsis thaliana FtsHi5 gene, and then transfects wild type Arabidopsis thaliana to obtain the FtsHi5 interference mutant, which shows yellowish dwarf, decreased chlorophyll content, and damaged thylakoid structure in chloroplast when DEX is added. Experiments have proved that the Arabidopsis FtsHi5 gene is involved in the regulation of chloroplast development and can be used to regulate chloroplast development.

Description of drawings

Fig. 1 is the identification figure of FtsHi5 interference strain; Wherein, WT: wild-type Arabidopsis Col-0; #6, #1, #2 represent the No. 6 strain of interference strain dex:RNAi-FtsHi5, 1 respectively No. strain and No. 2 strain; -DEX: not treated with dexamethasone (DEX) inducer; +DEX: treated with DEX inducer.

Fig. 2 is an analysis diagram of chlorophyll content; wherein, WT: wild-type Arabidopsis Col-0; #6, #1, #2 respectively represent No. 6 strain and No. 1 strain of the interference strain dex:RNAi-FtsHi5 , No. 2 strain; -DEX: not treated with dexamethasone (DEX) inducer; +DEX: treated with DEX inducer

Fig. 3 is an observation diagram of chloroplast development of FtsHi5 interference strain dex:RNAi-FtsHi5 No. 6 strain.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Wild-type Arabidopsis Columbia (Col-0) was purchased from Arabidopsis Biological Resource Center (http://www.arabidopsis.org/).

Embodiment 1: Construction and identification of FtsHi5 interference strain

1.1 Vector construction

1) Amplifying the FtsHi5 fragment of the target gene: using the cDNA of Col-0 as a template, amplifying the target fragment 1; the target fragment 1 is located at positions 157-587 of SEQ ID NO:1.

The primer sequences are as follows (5'→3'):

RNAi-FtsHi5-F: CGACTCTAGCCTCGAGTGCCCATTAGAGGCTGCTTT;

RNAi-FtsHi5-R:ACCAATTGGGGTACCGCAACCTCTCCATTTTCCTTTCT AACT;

The PCR reaction system is shown in Table 1:

Table 1 PCR reaction system (50μL)

components Dosage 2×Primer STAR Mix 25 μL RNAi-FtsHi5-F (10μM) 2.5μL RNAi-FtsHi5-R (10μM) 2.5μL cDNA 2μL ddH ₂ O 18μL

PCR amplification program:

98°C for 10s; 98°C for 10s, 55°C for 5s, 72°C for 1min/kb (26 cycles); 72°C for 10min; store at 4°C.

PCR product recovery: PCR products were run on 1% agarose gel, and the gel was cut and recovered. For specific methods, refer to the DNA recovery kit of Tiangen Company.

Using the cDNA of Col-0 as a template, RNAi-FtsHi5-F' and RNAi-FtsHi5-R' were used to amplify the reverse sequence of target fragment 1, which was designated as target fragment 2.

RNAi-FtsHi5-F': GAAATCGATAAGCTTGCAACCTCTCCATTTTCCTTTCTAACT;

RNAi-FtsHi5-R': GCCTGGATCGACTAGGTGCCCATTAGAGGCTGCTTT.

The PCR reaction system, amplification procedure and product recovery were carried out according to the above method.

2) Amplify the PDK intron fragment: use the PHANNIBAL vector (purchased from the Biovector Science Lab, China Plasmid Vector Strain Cell Line Gene Collection Center) as a template, use primer F and primer R to amplify the target fragment 3, and the primer sequence is as follows (5' →3′):

Primer F: GGTACCCCAATTGGTAAGGAAATAATT;

Primer R: AAGCTTATCGATTTCGAACCCAATTTCC;

The PCR reaction system is shown in Table 2:

Table 2 PCR reaction system

components Dosage 2×Primer STAR Mix 25 μL Primer F (10μM) 2.5μL Primer R (10μM) 2.5μL PHANNIBAL carrier 100ng ddH ₂ O Up to 50μL

PCR amplification program:

98°C 10s; 98°C 10s, 55°C 5s, 72°C 1min/kb (26 cycles); 72°C 10min; 4°C storage

PCR product recovery: PCR products were run on 1% agarose gel, and the gel was cut and recovered. For specific methods, refer to the DNA recovery kit of Tiangen Company.

Note: The above 2×Primer STAR Mix high-fidelity enzymes were purchased from Takara Company.

3) Double digestion linearized pTA7002-avrPto vector

The enzyme digestion reaction system is shown in Table 3:

Table 3 enzyme digestion reaction system

components Dosage 10×Buffer 3μL Enzyme1 (XhoI) 1μL Enzyme2 (SpeI) 1μL pTA7002-avrPto vector 1μg ddH2O Up to 30μL

Digestion conditions: 37°C, digest for 4 hours.

Recovery of digested products: After digested, add 3 μL of 10×Loading Buffer to the digested products and mix well. Electrophoresis was performed on 1% agarose gel, and the gel was cut for recovery. The recovery steps were performed according to the instructions of the DNA purification and recovery kit of Tiangen Company. The pTA7002-avrPto vector used was purchased from addgene company.

4) Construction of FtsHi5 interference vector by homologous recombination

The homologous recombination reaction system is shown in Table 4:

Table 4 reaction system

components Dosage Linearized pTA7002-avrPto vector X ng Destination Fragment 1 Y ng target fragment 2 M ng target fragment 3 W ng 5×CE∥Buffer 6μL Exnase∥ 3μL ddH ₂ O up to 30μL

Explanation: Optimal amount of carrier used X=[0.02×number of base pairs of cloning carrier]ng;

Optimal amount of fragments used Y=[0.04×number of base pairs of inserted fragments]ng

Optimal amount of fragment used M=[0.04×number of base pairs of inserted fragment]ng

Optimum fragment usage W=[0.04×number of base pairs of inserted fragment]ng.

Recombination reaction conditions: 37°C, 30min, put on ice for 5min. Store at -20°C.

Note: Recombinase Exnase∥ kit was purchased from Takara Company.

5) Transformation: Add the ligation product to Escherichia coli DH5α competent cells (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), mix gently, ice-bath for 20 minutes, heat shock at 42°C for 90 seconds, ice-bath again for 5 minutes, and add 400 μL Liquid without anti-LB, cultured at 37°C, 200rpm for 1h, took 200 μL and spread it on a Kanamycin-resistant (50 μg/mL) LB plate, and cultured overnight at 37°C.

6) Colony PCR detection: select a single colony for colony PCR detection to exclude false positive recombinants. Send the correctly detected single colonies to Sangon for sequencing. Correct sequencing indicates that the recombinant vector was constructed successfully, that is, the FtsHi5 interference vector was constructed.

7) Bacillus transformation

Add 1 μg of recombinant plasmid to Agrobacterium GV3101 competent cells (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), mix gently, ice bath for 30 minutes, liquid nitrogen quick freezing for 5 minutes, 37 ° C water bath for 2 minutes, add 500 μL liquid without antibiotic LB, cultured on a shaker at 100 rpm at 28°C for 3 hours; spread on a plate containing rifampicin (50 μg/mL), gentamicin (50 μg/mL) and kanamycin resistance (50 μg/mL), and cultured at 28°C for 2 hours sky.

1.2 Acquisition and screening of transgenic positive seedlings

1) Agrobacterium flower-dipping transformation After the recombinant plasmid was transformed into Agrobacterium GV3101, it was transferred to Arabidopsis thaliana by flower-dipping method. Inoculate a single colony of Agrobacterium containing the target gene into 3 mL of liquid LB containing the carrier resistance of the target gene, and incubate at 28°C and 200 rpm for 12 hours; Incubate in liquid LB resistant to damycin (50 μg/mL) and kanamycin (50 μg/mL) at 28°C and 200 rpm for 12 hours; collect the bacteria by centrifugation; resuspend the bacteria with Agrobacterium transformation solution and adjust to OD ₆₀₀ = 0.8-1.0; soak the Arabidopsis flowers in the transformation solution for 1 min; place the materials horizontally after treatment, and cultivate them in a dark environment for 24 hours.

2) Screening: The seeds of the transformed plants are the T1 generation, sow the T1 generation seeds on hygromycin (50 μg/mL) resistant MS medium (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), and in normal light Cultivate in an incubator for about 1 week, observe and screen positive seedlings. Transfer the well-grown T1 generation resistant seedlings to normal plant culture medium and cultivate them for 2 to 3 days, then transplant the resistant seedlings to nutrient soil (the nutrient soil is Jiffy peat soil imported from the Netherlands, purchased from Jiffy Company , website: https://www.jiffygroup.com/), after the seeds are mature, the single plant is harvested, and the T2 generation is obtained. The T2 generation was continuously screened in the MS medium containing hygromycin resistance until homozygous transgenic lines were obtained.

In order to study the effect of FtsHi5 gene on chloroplast development of Arabidopsis thaliana, FtsHi5 inducible interference lines were constructed in this experiment. A fragment (431bp) of the full-length cDNA of the FtsHi5 gene and the PDK (pyruvateorthophosphate dikinase) intron on the PHANNIBAL vector were selected and connected to the inducible vector pTA7002-avrPto by homologous recombination. The recombinant plasmid with correct sequencing was transformed into Agrobacterium GV3101, and the FtsHi5 interference vector was transformed into wild-type Arabidopsis Columbia (Col-0) by flower dipping method. Finally, a number of interference strains were obtained through screening, which were named dex:RNAi-FtsHi5 strain No. 1, dex:RNAi-FtsHi5 strain No. 2, and dex:RNAi-FtsHi5 strain No. 6, respectively abbreviated as #1 and # 2. #6. It was identified that the growth and development of the interfering strains were severely affected after being treated with dexamethasone (DEX, the concentration was 5 μmol/L) inducer for 7 days, and the transcription level of FtsHi5 was down-regulated to varying degrees (Fig. 1A), and the corresponding yellowing phenotype appeared in the leaves ( Figure 1B). However, in the culture medium without adding inducer (control group), there was no significant difference between the interference strain dex:RNAi-FtsHi5 and the wild type.

Embodiment 2: the mensuration of chlorophyll content and chloroplast ultrastructure observation

The wild-type Arabidopsis Col-0 and the interference line dex:RNAi-FtsHi5 (#1, #2, #6) grown for 2 weeks in the MS medium induced by adding and not adding DEX (concentration: 5 μmol/L) ) of chlorophyll content. It can be seen from Figure 2 that by measuring the chlorophyll content of wild-type Arabidopsis Col-0 and the interference strain dex:RNAi-FtsHi5 (#1, #2, #6) when adding and not adding DEX, it was found that FtsHi5 was destroyed or down-regulated. Transcript expression, leaf chlorophyll content decreased. As can be seen from Figure 3, the chloroplast ultrastructure of dex:RNAi-FtsHi5#6 plants was observed by transmission electron microscopy. Compared with the control group, it was found in the chloroplasts of the interference plants induced by DEX (30 μmol/L) for 7 days and 14 days After down-regulating the transcription and expression of FtsHi5, the endocyst structure was damaged, which further indicated that FtsHi5 gene was involved in the regulation of chloroplast development.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

<120> Application of Arabidopsis thaliana FtsHi5 Gene in Regulation of Arabidopsis Chloroplast Development

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3963

<212>DNA

<213> Artificial Sequence

<220>

<223> FtsHi5 gene coding sequence

<400> 1

atggatttca tttccgcttc ttccttatct tcaccctttt ccacacagtt gtctccgatt 60

tatttgagct caggtatagt atcgttaaag cctagacatc gagtcaaaaa tcgaaacttt 120

gggtctagag aatccaataa taaatctagg aaaattgtgc ccattagagg ctgctttggt 180

ttttccggga gttttttgag gtcgaaacag tctgattacg gtagtgaagc tgtttcggaa 240

tcattgagat tatgtggaga gggaaatgag cttgtgctat cttccgagta taatagcgca 300

aagacgagag agagcgtaat acagtttgtt accaagcctc tagtttacgc cttgttttgt 360

attgcaattg gactttctcc gattcgctct tttcaagctc cagctttagc tgtgcctttc 420

gtcagtgatg ttatttggaa gaaaaagaaa gagagagtga gagagaaaga ggtggttttg 480

aaagcagttg atcatgagtt ctcggattat actcggagat tgttggagac agtctcggtt 540

ttactgaaga ctattgagat agttagaaag gaaaatggag aggttgctga agtgggagcg 600

gctttggatg cagttaaggt ggagaaggag aaactgcaga aggagataat gagtggggtta 660

tatcgtgata tgaggcggtt gaggaaggag agagatttgt taatgaaacg agctgataaa 720

attgtagatg aggccctaag tttgaagaaa cagagtgaaa aactgttgag aaaaggtgct 780

agggagaaaa tggagaaact ggaagagagt gtggatatta tggagagcga gtacaataag 840

atatgggaaa gaatagatga gattgatgat atcattttaa agaaagagac aacaacattg 900

agttttgggg ttaggggagct tattttcatt gaaagagagt gtgtagagtt ggttaagagc 960

ttcaaccgag aattgaatca aaaaagtttt gaaagtgttc ctgaaagctc cattacaaaa 1020

ctctcgaggt ctgagattaa gcaggaatta gtgaacgctc aaagaaagca cttggaacaa 1080

atgatactgc ccaatgtttt ggaactagag gaggttgatc cctttttcga tcgtgactca 1140

gtggattttt ctctacgcat caaaaaacgc cttgaggaat caaaaaagct gcagagagat 1200

cttcagaatc gtatcagaaa acgtatgaaa aagtttggtg aagaaaagct ttttgttcaa 1260

aagactccag aaggtgaagc tgtcaaggga tttcctgaag cagaggtgaa atggatgttt 1320

ggagagaaag aggtggttgt tccgaaagct atccagctcc atctacgcca tggttggaaa 1380

aagtggcagg aagaagcaaa agccgatta aaacagaaac ttttggaaga tgttgatttt 1440

ggcaaacaat atattgcgca gagacaggaa caagttctcc tggatcgaga tagagttgtt 1500

tcgaagactt ggtataacga agacaaaagt aggtgggaga tggaccctat ggctgttccc 1560

tacgcagttt ccaggaagct gattgacagt gccaggatta ggcatgatta tgctgtaatg 1620

tatgttgcat taaaaggaga tgacaaggag ttttacgttg atatcaagga atatgagatg 1680

ctatttgaga aatttggagg gtttgatgct ctgtacctca aaatgcttgc ttgtggaatc 1740

ccaacttctg ttcatctgat gtggataccc atgtcagagc taagtcttca acaacagttt 1800

ctgttggtta caagggtggt ttctcgagtc tttaacgcat tgaggaagac tcaaggttgta 1860

tcaaatgcga aggatacagt attagagaaa ataaggaata taaacgatga cattatgatg 1920

gcggttgtgt ttccagttat cgaatttatc attccttacc agttaaggct gcgtctggga 1980

atggcttggc cagaagaaat agaacagact gttggctcaa catggtactt acaatggcag 2040

tctgaagcag aaatgaattt caagtcccgc aatacagaag attttcagtg gttcctctgg 2100

tttctaattc gaagttctat atacgggttt gttttgtatc acgttttccg ctttctaaaa 2160

agaaaggttc caagacttct tggctatgga cccttccgcc gagatcccaa cgtacggaaa 2220

ttctggagag tgaaatcata tttcacttat agaaaaagga gaatcaaaca aaagagaaag 2280

gctggaatcg acccccataaa aactgcattc gacagaatga agagagtgaa aaatccacct 2340

atcccattaa agaactttgc tagtattgag tctatgagag aagaaatcaa cgaggttgtg 2400

gcatttttac agaatccaaa ggcatttcag gagatggggag cccgcgcacc tcggggcgtt 2460

cttattgtgg gtgagagagg aacgggaaag acgtcactgg cgctcgctat agcagccgaa 2520

gcaagagtgc ctgttgtcaa tgttgaagct caagagttgg aagctgggct atgggttggt 2580

caaagtgcag cgaatgtcag ggaactcttt caaactgcaa gagacttggc acctgtaatc 2640

atttttgtgg aggattttga cctctttgct ggtgtacgcg ggaagtttgt acatacaaaa 2700

cagcaagatc atgaatcgtt cattaatcag cttcttgttg aacttgatgg ctttgagaaa 2760

caggatggcg tagttttgat ggcgacgaca cgaaatcata agcaaattga tgaggcgcta 2820

agaaggccag gtcgtatgga tagagtattc catcttcaaa gtccaactga aatggaaagg 2880

gagaggattt tacacaacgc tgcagaagaa accatggacc gagaactcgt tgatctagtg 2940

gattggcgta aggtttcaga gaagacaact ttattaagac cgatagaact gaaacttgtt 3000

ccaatggctc tcgaaagtag tgccttcaga agcaaatttc tggacactga cgaacttctg 3060

agttacgtta gctggtttgc gacgtttagc catatagtgc ccccatggct gcgaaaaact 3120

aaagttgcaa agacaatggg caaaatgctt gtgaatcatc ttggacttaa cttaaccaaa 3180

gatgatttgg agaatgtggt tgatctgatg gaaccatatg gacaaataag taatggaata 3240

gaacttttga atcctactgt tgattggacg agggagacaa agttccccaca tgctgtctgg 3300

gcggctggtc gtgctctcat cacacttcta ataccaaact ttgatgttgt ggaaaatctt 3360

tggcttgagc ctagttcctg ggaaggaatt gggtgtacaa aaatcaccaa ggtcacaagt 3420

ggaggctccg cgattgggaa cacggaatca agatcatatc ttgaaaagaa gctcgttttc 3480

tgctttggat ctcacattgc atcccaaatg cttcttcctc ccggagatga aaattttctt 3540

tcttcttcag aaataactaa agcgcaggag attgcaacac gtatggtgct tcaatatggc 3600

tggggacccg atgacagtcc tgcagtatac tatgctacta acgcggtttc agctttgagt 3660

atggggaaca atcacgaata tgaaatggcg ggtaaagttg aaaagatta tgacttagcg 3720

tacgaaaagg caaagggaat gctccttaaa aatcgacgtg tccttgagaa aatcacagag 3780

gagctactcg aatttgagat tttaactcat aaggatttgg aaagaattgt tcatgagaat 3840

ggtgggattc gggagaagga acctttcttt ctatcaggaa ccaattacaa tgaggcattg 3900

tcaaggagtt ttcttgatgt tggagatccg ccagaaactg cacttcttag tgcaccaacc 3960

taa 3963

<210> 2

<211> 1320

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of FtsHi5 gene

<400> 2

Met Asp Phe Ile Ser Ala Ser Ser Leu Ser Ser Pro Phe Ser Thr Gln

1 5 10 15

Leu Ser Pro Ile Tyr Leu Ser Ser Gly Ile Val Ser Leu Lys Pro Arg

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His Arg Val Lys Asn Arg Asn Phe Gly Ser Arg Glu Ser Asn Asn Lys

35 40 45

Ser Arg Lys Ile Val Pro Ile Arg Gly Cys Phe Gly Phe Ser Gly Ser

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Phe Leu Arg Ser Lys Gln Ser Asp Tyr Gly Ser Glu Ala Val Ser Glu

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Ser Leu Arg Leu Cys Gly Glu Gly Asn Glu Leu Val Leu Ser Ser Ser Glu

85 90 95

Tyr Asn Ser Ala Lys Thr Arg Glu Ser Val Ile Gln Phe Val Thr Lys

100 105 110

Pro Leu Val Tyr Ala Leu Phe Cys Ile Ala Ile Gly Leu Ser Pro Ile

115 120 125

Arg Ser Phe Gln Ala Pro Ala Leu Ala Val Pro Phe Val Ser Asp Val

130 135 140

Ile Trp Lys Lys Lys Lys Lys Glu Arg Val Arg Glu Lys Glu Val Val Leu

145 150 155 160

Lys Ala Val Asp His Glu Phe Ser Asp Tyr Thr Arg Arg Leu Leu Glu

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Thr Val Ser Val Leu Leu Lys Thr Ile Glu Ile Val Arg Lys Glu Asn

180 185 190

Gly Glu Val Ala Glu Val Gly Ala Ala Leu Asp Ala Val Lys Val Glu

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Lys Glu Lys Leu Gln Lys Glu Ile Met Ser Gly Leu Tyr Arg Asp Met

210 215 220

Arg Arg Leu Arg Lys Glu Arg Asp Leu Leu Met Lys Arg Ala Asp Lys

225 230 235 240

Ile Val Asp Glu Ala Leu Ser Leu Lys Lys Gln Ser Glu Lys Leu Leu

245 250 255

Arg Lys Gly Ala Arg Glu Lys Met Glu Lys Leu Glu Glu Ser Val Asp

260 265 270

Ile Met Glu Ser Glu Tyr Asn Lys Ile Trp Glu Arg Ile Asp Glu Ile

275 280 285

Asp Asp Ile Ile Leu Lys Lys Glu Thr Thr Thr Leu Ser Phe Gly Val

290 295 300

Arg Glu Leu Ile Phe Ile Glu Arg Glu Cys Val Glu Leu Val Lys Ser

305 310 315 320

Phe Asn Arg Glu Leu Asn Gln Lys Ser Phe Glu Ser Val Pro Glu Ser

325 330 335

Ser Ile Thr Lys Leu Ser Arg Ser Glu Ile Lys Gln Glu Leu Val Asn

340 345 350

Ala Gln Arg Lys His Leu Glu Gln Met Ile Leu Pro Asn Val Leu Glu

355 360 365

Leu Glu Glu Val Asp Pro Phe Phe Asp Arg Asp Ser Val Asp Phe Ser

370 375 380

Leu Arg Ile Lys Lys Arg Leu Glu Glu Ser Lys Lys Leu Gln Arg Asp

385 390 395 400

Leu Gln Asn Arg Ile Arg Lys Arg Met Lys Lys Phe Gly Glu Glu Lys

405 410 415

Leu Phe Val Gln Lys Thr Pro Glu Gly Glu Ala Val Lys Gly Phe Pro

420 425 430

Glu Ala Glu Val Lys Trp Met Phe Gly Glu Lys Glu Val Val Val Pro

435 440 445

Lys Ala Ile Gln Leu His Leu Arg His Gly Trp Lys Lys Trp Gln Glu

450 455 460

Glu Ala Lys Ala Asp Leu Lys Gln Lys Leu Leu Glu Asp Val Asp Phe

465 470 475 480

Gly Lys Gln Tyr Ile Ala Gln Arg Gln Glu Gln Val Leu Leu Asp Arg

485 490 495

Asp Arg Val Val Ser Lys Thr Trp Tyr Asn Glu Asp Lys Ser Arg Trp

500 505 510

Glu Met Asp Pro Met Ala Val Pro Tyr Ala Val Ser Arg Lys Leu Ile

515 520 525

Asp Ser Ala Arg Ile Arg His Asp Tyr Ala Val Met Tyr Val Ala Leu

530 535 540

Lys Gly Asp Asp Lys Glu Phe Tyr Val Asp Ile Lys Glu Tyr Glu Met

545 550 555 560

Leu Phe Glu Lys Phe Gly Gly Phe Asp Ala Leu Tyr Leu Lys Met Leu

565 570 575

Ala Cys Gly Ile Pro Thr Ser Val His Leu Met Trp Ile Pro Met Ser

580 585 590

Glu Leu Ser Leu Gln Gln Gln Phe Leu Leu Val Thr Arg Val Val Ser

595 600 605

Arg Val Phe Asn Ala Leu Arg Lys Thr Gln Val Val Ser Asn Ala Lys

610 615 620

Asp Thr Val Leu Glu Lys Ile Arg Asn Ile Asn Asp Asp Ile Met Met

625 630 635 640

Ala Val Val Phe Pro Val Ile Glu Phe Ile Ile Pro Tyr Gln Leu Arg

645 650 655

Leu Arg Leu Gly Met Ala Trp Pro Glu Glu Ile Glu Gln Thr Val Gly

660 665 670

Ser Thr Trp Tyr Leu Gln Trp Gln Ser Glu Ala Glu Met Asn Phe Lys

675 680 685

Ser Arg Asn Thr Glu Asp Phe Gln Trp Phe Leu Trp Phe Leu Ile Arg

690 695 700

Ser Ser Ile Tyr Gly Phe Val Leu Tyr His Val Phe Arg Phe Leu Lys

705 710 715 720

Arg Lys Val Pro Arg Leu Leu Gly Tyr Gly Pro Phe Arg Arg Asp Pro

725 730 735

Asn Val Arg Lys Phe Trp Arg Val Lys Ser Tyr Phe Thr Tyr Arg Lys

740 745 750

Arg Arg Ile Lys Gln Lys Arg Lys Ala Gly Ile Asp Pro Ile Lys Thr

755 760 765

Ala Phe Asp Arg Met Lys Arg Val Lys Asn Pro Pro Ile Pro Leu Lys

770 775 780

Asn Phe Ala Ser Ile Glu Ser Met Arg Glu Glu Ile Asn Glu Val Val

785 790 795 800

Ala Phe Leu Gln Asn Pro Lys Ala Phe Gln Glu Met Gly Ala Arg Ala

805 810 815

Pro Arg Gly Val Leu Ile Val Gly Glu Arg Gly Thr Gly Lys Thr Ser

820 825 830

Leu Ala Leu Ala Ile Ala Ala Glu Ala Arg Val Pro Val Val Asn Val

835 840 845

Glu Ala Gln Glu Leu Glu Ala Gly Leu Trp Val Gly Gln Ser Ala Ala

850 855 860

Asn Val Arg Glu Leu Phe Gln Thr Ala Arg Asp Leu Ala Pro Val Ile

865 870 875 880

Ile Phe Val Glu Asp Phe Asp Leu Phe Ala Gly Val Arg Gly Lys Phe

885 890 895

Val His Thr Lys Gln Gln Asp His Glu Ser Phe Ile Asn Gln Leu Leu

900 905 910

Val Glu Leu Asp Gly Phe Glu Lys Gln Asp Gly Val Val Leu Met Ala

915 920 925

Thr Thr Arg Asn His Lys Gln Ile Asp Glu Ala Leu Arg Arg Pro Gly

930 935 940

Arg Met Asp Arg Val Phe His Leu Gln Ser Pro Thr Glu Met Glu Arg

945 950 955 960

Glu Arg Ile Leu His Asn Ala Ala Glu Glu Thr Met Asp Arg Glu Leu

965 970 975

Val Asp Leu Val Asp Trp Arg Lys Val Ser Glu Lys Thr Thr Leu Leu

980 985 990

Arg Pro Ile Glu Leu Lys Leu Val Pro Met Ala Leu Glu Ser Ser Ala

995 1000 1005

Phe Arg Ser Lys Phe Leu Asp Thr Asp Glu Leu Leu Ser Tyr Val Ser

1010 1015 1020

Trp Phe Ala Thr Phe Ser His Ile Val Pro Pro Trp Leu Arg Lys Thr

1025 1030 1035 1040

Lys Val Ala Lys Thr Met Gly Lys Met Leu Val Asn His Leu Gly Leu

1045 1050 1055

Asn Leu Thr Lys Asp Asp Leu Glu Asn Val Val Asp Leu Met Glu Pro

1060 1065 1070

Tyr Gly Gln Ile Ser Asn Gly Ile Glu Leu Leu Asn Pro Thr Val Asp

1075 1080 1085

Trp Thr Arg Glu Thr Lys Phe Pro His Ala Val Trp Ala Ala Gly Arg

1090 1095 1100

Ala Leu Ile Thr Leu Leu Ile Pro Asn Phe Asp Val Val Glu Asn Leu

1105 1110 1115 1120

Trp Leu Glu Pro Ser Ser Trp Glu Gly Ile Gly Cys Thr Lys Ile Thr

1125 1130 1135

Lys Val Thr Ser Gly Gly Ser Ala Ile Gly Asn Thr Glu Ser Arg Ser

1140 1145 1150

Tyr Leu Glu Lys Lys Leu Val Phe Cys Phe Gly Ser His Ile Ala Ser

1155 1160 1165

Gln Met Leu Leu Pro Pro Gly Asp Glu Asn Phe Leu Ser Ser Ser Ser Glu

1170 1175 1180

Ile Thr Lys Ala Gln Glu Ile Ala Thr Arg Met Val Leu Gln Tyr Gly

1185 1190 1195 1200

Trp Gly Pro Asp Asp Ser Pro Ala Val Tyr Tyr Ala Thr Asn Ala Val

1205 1210 1215

Ser Ala Leu Ser Met Gly Asn Asn His Glu Tyr Glu Met Ala Gly Lys

1220 1225 1230

Val Glu Lys Ile Tyr Asp Leu Ala Tyr Glu Lys Ala Lys Gly Met Leu

1235 1240 1245

Leu Lys Asn Arg Arg Val Leu Glu Lys Ile Thr Glu Glu Leu Leu Glu

1250 1255 1260

Phe Glu Ile Leu Thr His Lys Asp Leu Glu Arg Ile Val His Glu Asn

1265 1270 1275 1280

Gly Gly Ile Arg Glu Lys Glu Pro Phe Phe Leu Ser Gly Thr Asn Tyr

1285 1290 1295

Asn Glu Ala Leu Ser Arg Ser Phe Leu Asp Val Gly Asp Pro Pro Glu

1300 1305 1310

Thr Ala Leu Leu Ser Ala Pro Thr

1315 1320

<210> 3

<211> 36

<212>DNA

<213> Artificial Sequence

<220>

<223> RNAi-FtsHi5-F

<400> 3

cgactctagc ctcgagtgcc cattagaggc tgcttt 36

<210> 4

<211> 42

<212>DNA

<213> Artificial Sequence

<220>

<223> RNAi-FtsHi5-R

<400> 4

accaattggg gtaccgcaac ctctccattt tcctttctaa ct 42

<210> 5

<211> 42

<212>DNA

<213> Artificial Sequence

<220>

<223> RNAi-FtsHi5-F'

<400> 5

gaaatcgata agcttgcaac ctctccattt tcctttctaa ct 42

<210> 6

<211> 36

<212>DNA

<213> Artificial Sequence

<220>

<223> RNAi-FtsHi5-R'

<400> 6

gcctggatcg actaggtgcc cattagaggc tgcttt 36

<210> 7

<211> 27

<212>DNA

<213> Artificial Sequence

<220>

<223> Primer F

<400> 7

ggtaccccaa ttggtaagga aataatt 27

<210> 8

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<223> Primer R

<400> 8

aagcttatcg atttcgaacc caatttcc 28

Claims (6)

1. An application of Arabidopsis thaliana FtsHi5 gene in regulating the development of Arabidopsis chloroplasts. 2. Application according to claim 1, characterized in that: Application of the Arabidopsis FtsHi5 gene in plant breeding. 3. The application according to claim 1, characterized in that: Application of the Arabidopsis FtsHi5 gene in breeding transgenic plants. 4. The application according to claim 2 or 3, characterized in that: Said plants include Arabidopsis, rice, wheat, corn, sorghum, millet, sugar cane, cotton, tomato, alfalfa and elephant grass. 5. The application according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the Arabidopsis FtsHi5 gene is shown in SEQ ID NO:2. 6. The application according to any one of claims 1 to 3, characterized in that: The sequence of the Arabidopsis FtsHi5 gene is one of the following nucleotide sequences: 1) The DNA sequence shown in SEQ ID NO: 1 in the sequence listing; 2) The DNA sequence of the protein encoded by SEQ ID NO: 1 in the sequence listing.