水稻丝氨酸棕榈酰转移酶的分子克隆、特征及其与褐飞虱抗性相关的基因表达

DOI :10.3785/j.issn.1008-9209.2017.11.211
Molecular cloning and characterization of a serine palmitoyltransferase gene from
rice (Oryza sativa )and its gene expression in defense response to brown planthopper
BEGUM Mahfuj Ara 1,SHI Xiaoxiao 1,BAI Yueliang 1,JIANG Yandong 1,ZHOU Wenwu 1,MAO Cungui 2,ZHU Zengrong 1*(1.Institute of Insect Sciences,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China;2.Department of Medicine and Stony Brook Cancer Center,The State University of New York at Stony Brook,New York 11794,USA )Abstract To understand potential mechanisms involved in sphingolipids and their metabolic precursors,long-chain bases (LCBs)-mediated stress response,we cloned rice LCB genes that encode serine palmitoyltransferase (SPT),which is the key enzyme of sphingolipid de novo biosynthesis.The full-length cDNAs of three LCB1and three LCB2genes were amplified by reverse transcription -polymerase chain reaction (RT -PCR).The lengths of the deduced LCB proteins ranged from 481to 497amino acid residues.Bioinformatics and comparative studies revealed that the polypeptide proteins had high homology in a large number of species from bacteria to humans.In rice,we found that OsLCB2a1expression was up-regulated when infested by brown planthopper (BPH).Compared with other varieties,gene expression levels were higher in BPH-resistant varieties such as Mudgo and IR64.Gene expression was negatively correlated with BPH-resistant scores.Among different plant parts of rice varieties,gene expression was found to be significantly increased in the leaf blade of IR64and the leaf sheath of Taiping.The mRNA level of OsLCB2a1was found to be high at the maximum tillering stage of BPH-resistant varieties,suggesting that this gene may play a role in the vegetative stages of rice.
Key words sphingolipid metabolism;serine palmitoyltransferase;defense;gene expression pattern CLC number S 186;Q 78Document code
A
水稻丝氨酸棕榈酰转移酶的分子克隆、特征及其与褐飞虱抗性相关的基因表达(英文).浙江大学学报
（农业与生命科学版）,2018,44(3):365-372
BEGUM Mahfuj Ara 1，史肖肖1，白月亮1，蒋艳冬1，周文武1，毛存贵2，祝增荣1*（1.浙江大学农业与生物技术学院昆虫科学研究所，杭州310058；2.美国纽约州立大学石溪分校医学系和石溪癌症中心，纽约11794）摘要
为研究鞘脂质和鞘脂质代谢前体长链基团（long-chain bases,LCBs ）对胁迫应答的调控机制，对编码水稻丝
氨酸棕榈酰转移酶（serine palmitoyltransferase,SPT ）的LCB 基因进行克隆。

通过反转录聚合酶链式反应（reverse transcription -polymerase chain reaction,RT -PCR ）扩增到3条LCB1和3条LCB2基因全长，蛋白质大小为481～497个氨基酸。

生物信息学分析表明，LCB 蛋白序列在细菌到人等多个物种中具有高度保守性。

在褐飞虱（brown planthopper,BPH ）的侵害作用下，水稻OsLCB2a1基因表达量上升。

与其他水稻品种相比，抗性品种“Mudgo ”和
Foundation item:Projects supported by the National Natural Science Foundation of China (31572001),Industry Project of the Ministry of Agriculture of China (201403030),and the National Key Basic Research and Development Project (973Program)(2010CB126200).*Corresponding author:ZHU Zengrong (https:///0000-0002-3247-1486),Tel:+86-571-88982355,E-mail:zrzhu@ First author:BEGUM Mahfuj Ara (https:///0000-0003-2073-5111),E-mail:mahfuj291@ Received:2017-11-21;Accepted :2018-05-02
浙江大学学报（农业与生命科学版）
44（3）：365～372，2018
Journal of Zhejiang University (Agric.&Life Sci.)
/agr E -mail:zdxbnsb @
Sphingolipids are major structural components of endomembranes and dynamic regulators of basic cellular processes in all eukaryotes and a few prokaryotes [1-6].The biosynthesis of sphingolipids is initiated in endoplasmic reticulum (ER)with the condensation of serine and fatty acyl -CoA.In particular,sphingolipids play a crucial role in divergent signaling events,including differentiation,senescence,proliferation,apoptosis,programmed cell death (PCD)and stress responses [7-12]
.Recent studies have
revealed
that
sphingolipids
also
showed
antimicrobial activity [13].Long-chain bases (LCBs)are metabolic precursors of complex sphingolipids.Serine palmitoyltransferase (SPT)is the key enzyme of sphingolipids de novo biosynthesis.This enzyme catalyzes the first reaction in LCB synthesis from the condensation of serine and palmitoyl -CoA to form 3-ketosphinganine.SPT is the rate-limiting step for biosynthesis of sphingolipids,which comprises three
subunits,
LCB1,LCB2a
and
LCB2b [14].
Disruption
of
SPT
in
Nicotiana
benthamiana
activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f.sp.lycopersici [15].Plants have evolved a complex innate immune system that can effectively protect plants against various biotic stresses.It is believed that individual plant cells have the capacity to detect a pathogen attack and to activate onsite defense responses.
In
fact,
sphingolipids
as
bioactive
molecules have been shown to be extensively involved in plant defense-associated programmed cell death (PCD),and more recently,sphingolipids have been
implicated
in
regulation of membrane trafficking
and/or
formation
of
membrane
subdomains during defense responses [16].Recent evidence suggests that the OsLCB2a1subunit of SPT
is involved in plant defense against herbivores [17].
In this study,we cloned SPT gene from rice.The transcription profiles of one SPT gene,OsLCB2a1,under different levels of brown planthopper (BPH)infestation were analyzed by quantitative real-time polymerase chain reaction (qRT -PCR).
1Materials and methods
1.1
Plant growth
The rice genotype Xiushui 11(Oryza sativa japonica variety)was used for gene cloning.Eight varieties (Xiushui 11,IR64,Taiping,HybridSy63,Mudgo,Wuyugen2,Guanglu ’ai and TN1)were used for varietal expression study.Seeds were reared in a plastic box containing rice nutrient solution [18],and maintained in a controlled climate room at (26±2)℃,12h light phase and 80%relative humidity.All the collected samples were immediately frozen in liquid nitrogen and maintained at －80℃for RNA extractions.There were five replications for each treatment.1.2
Insects
Colonies of the brown planthopper (Nilaparvata lugens )were maintained on Taichung Native 1(TN1,an indica variety without any resistant gene to herbivores and pathogens)rice seedlings in a controlled climate room at (26±2)℃,12h light phase and 80%relative humidity.1.3
Sequence analysis
We performed PCR reaction (Toyobo,Japan)to amplify the specific genes using primers in Table 1.PCR products were sequenced by a commercial company (Sunny,China).SPT homolog sequences were obtained by BLASTp (https://)and Phytozome 10.3(https://),and the representative sequences were aligned by
“IR64”中的基因表达量更高。

基因表达量与褐飞虱危害指数呈负相关。

在不同水稻品种的不同组织中，“IR64”叶片中的基因表达量显著升高，而“态平籼”叶鞘中的基因表达量显著升高。

OsLCB2a1基因在抗褐飞虱水稻品种的最高分蘖期呈高表达，表明该基因可能在水稻营养期发挥作用。

关键词鞘脂质代谢；丝氨酸棕榈酰转移酶；防御；基因表达模式
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CLUSTALW using GENETYX program(Software Development Co.Ltd.,Japan).The aligned sequences were used for phylogenetic analysis with neighbor-joining(NJ)method by MEGA6.0and bootstrapping for1000replicates[19].Accession numbers of different organisms used in this tree contraction from the
NCBI and Phytozome were given in Table S1(http:// /agr/EN/abstract/abstract30409. shtml).
1.4Statistical analysis
Data were analyzed using analysis of variance (ANOV A)and presented as mean values for each treatment.Statistical analysis was performed using Data Processing System(DPS)statistical software package[20-21].The Tukey’s test(P＜0.05)was performed to evaluate the treatment effects.
1.5Quantitative RT-PCR(qRT-PCR)
The total RNA from leaf or seedling samples was extracted using a extraction kit(Tiangen,Shanghai)and reversely transcribed to cDNA using a Prime Script RT reagent kit(Takara)according to the manufacture’s instruction.Gene-specific primers(Table2)were designed with Primer-3-Plus software.qRT-PCR was performed using SYBR Green Supermix Reagent (BioRad,USA),and run on an ABI7500Real-Time PCR System for one cycle of95℃for3min, followed by40cycles of95℃for10s,and60℃for 30s.Normalized gene expression was calculated by the 2－ΔΔC T method[22].1.6Evaluation for plant resistance against BPH
A seedling bulk test was conducted to evaluate the BPH resistance of rice varieties following previously described methods[23].Seeds were randomly sown in a plastic box in three26cm-long rows,with2.5cm between rows.The varieties IR64 and TN1were used as resistant and susceptible check,respectively.At the third-leaf stage,the seedlings were infested with the second to third-instar nymphs of BPH with10insects per seedling.When all of the seedlings of susceptible control(scored as 9)died,the plants of other rice were examined and each seedling was given a score of1to9according to the method[23];the lower scores indicate higher resistance to the insect.
2Results
2.1Cloning of the SPT genes
To investigate the possible function of SPT in insect resistance response,we cloned genes from O. sativa encoding the LCB1,LCB2and the subunit of SPT.The full-length cDNA of three LCB1and three LCB2genes were amplified by RT-PCR.
The lengths of the deduced LCB proteins ranged from481to497amino acid residues.To identify LCB homology,we used BLASTp to conduct sequence similarity searches using the sequence of Arabidopsis thaliana(AtLCB1,AT4G36480)and A. thaliana(AtLCB2a,AT5G23670).The results of the BLASTX search are shown in Table 3.Sequence alignment showed that LCB1.1had the highest identity(89%)to the LCB protein found in Sorghum bicolor.LCB1.2showed the highest identity(88%)to the serine palmitoyltransferase1found in Zea mays. LCB1.3showed the highest identity(94%)to the Table2Primers used in qRT-PCR assays Primer name
70-F
70-R
OsActinF
OsActinR
Primer sequence(5′—3′)
TCTATCTCCGCATCCAGGAC
ACACGAGGGGTGCAGTATTC
CAGCACATTCCAGCAGAT
GGCTTAGCATTCTTGGGT
Table1Primers used in PCR assays
Primer name SPT1F SPT1R SPT2F SPT2R SPT3F SPT3R SPT4F SPT4R SPT5F SPT5R SPT6F SPT6R
Primer sequence(5′—3′) ATGGAGATGGTGTTGCCAGT CTAGAGCACTGATGCTGCAACC ATGGAAATGGTATTGCCAGTCG CTAGACAACTGATGCTGCAACCT ATGGACATGGCATTGCCAAT TCAGATGTAATCTGACAGAA ATGGTGAGGCTGCCGTACA TTACTCCAGCTTATCGTGATCAG ATGGTGAGGCTGCCCTACGTGA TTGAAGCTCTTCAGTTTCTCAACAG ATGGTTCGGCTCGCCTACGGAA TTGAAACTCCCAGTTTCTCAGTCG
第3期BEGUM Mahfuj Ara，等：水稻丝氨酸棕榈酰转移酶的分子克隆、特征及其与褐飞虱抗性相关的基因表达(英文)367
A
B
Most conserved region is highlighted,and single asterisk (*)indicates no exact match amino acid residues.
Fig.1Multiple alignment of deduced amino acid sequences of LCB1(A)and LCB2(B)genes in different species
LCB found in Dichanthelium oligosanthes .On the other hand,LCB2a.1and LCB2a.2showed high identity to the LCB protein in Z.mays ,with 96%and 88%,respectively;whereas LCB2a.3showed the highest identity (97%)to LCB protein in S.bicolor .
Most conserved regions of LCB1and LCB2in different species were showed in multiple alignment (Fig.1A -B).
To investigate the evolutionary relationships,phylogenetic analysis was performed with the LCB
Table 3
BLASTX results of SPT genes in Oryza sativa
Gene name LCB1.1LCB1.2LCB1.3LCB2a.1LCB2a.2LCB2a.3Accession No.XP_015622652.1XP_015612907.1XP_015633207.1XP_015613397.1XP_015613384.1XP_015617901.1
aa No.481482485489497488
BLASTX best hit
Name
Long chain base
biosynthesis protein 1a Serine palmitoyltrans-ferase 1
Long chain base
biosynthesis protein 1a Long chain base
biosynthesis protein 2d Long chain base
biosynthesis protein 2d Long chain base
biosynthesis protein 2a
Species Sorghum bicolor Zea mays Dichanthelium oligosanthes Zea mays Zea mays Sorghum bicolor
Accession No.XP_002465548NP_001147940OEL38014NP_001295432XP055613384XP_002450805
E -value 0.00.00.00.00.00.0
Identity/%
898894968897
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protein from O.sativa and other species (Fig.2).The NJ tree revealed that three LCB1genes clustered with one sub-branch had very closely related homologues with LCB of Brachypodium distachyon and Z.mays .Moreover,we found that two LCB2a genes were clustered with B.distachyon in one single branch,and had very closely related homologues with Z.mays and S.bicolor .On the other hand,another LCB2a gene clustered in separate clad had very closely related homologues with Z.mays and B.distachyon .The accession number of proteins were listed in Table S1(/agr/EN/abstract/abstract30409.shtml).
2.2Expression analysis of SPT in different levels of BPH infestation
When we subjected rice plants to BPH feeding,the
results showed that OsLCB2a1transcripts increased at 4h after infestation and then gradually decreased from 8to 24h (Fig.3).Plants subjected the physical wound had much lower expressions.
We also studied OsLCB2a1gene expression in different varieties,different plant parts and growth stages of rice.Among eight different rice varieties (Xiushui 11,Wuyugen2,IR64,Taiping,HybridSy63,Mudgo,Guanglu ’ai and TN1,all are indica varieties except the first two),the gene expression levels were
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OsLCB1.1,OsLCB1.2,OsLCB1.3are three gene isoforms of OsLCB1;OsLCB2a.1,OsLCB2a.2,OsLCB2a.3are three gene isoforms of OsLCB2a .Rice sequences are marked with red dot.At:Arabidopsis thaliana ;Ao:Aspergillus oryzae ;Bd :Brachypo diumdistachyon ;Cr:Clamydomonas reinhardtii ;Cs:Citrus sinensis ;Dm:Drosophila melanogaster ;Eg:Eucalyptus grandis ;Gm:Glycine max ;Hs:Homo sapiens ;Ms:Mus musculus ;Os:Oryza sativa ;P:Pseudomonas sp.;Pp:Physcomitrella patens ;Pt:Populus trycocarpa ;Sb:Sorghum bicolor ;Sm:Selaginella moellendorffii ;Sl:Solanum lycopersicum ;Vv:Vitis vinifera ;Zm:Zea mays .
Fig.2Phylogenetic relationships of serine palmitoyltransferase protein (LCB1and LCB2)from various species
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369
significantly higher in Taiping (P ＜0.05)and lower in TN1,a susceptible rice variety for BPH;it was also high in BPH-resistant varieties like Mudgo and IR64,but was low in the other four varieties (Fig.4A).Gene expression was negatively correlated with BPH resistance scores (Fig.4B).
Gene expression of OsLCB2a1was determined in leaf blade,leaf sheath and roots of three rice varieties,
i.e.IR64,Taiping and TN1.Among different plant parts
of rice varieties,gene expression was found to be significantly higher in the leaf blade of IR64and the leaf sheath of Taiping,and comparatively lower in the roots compared with the leaf blade and leaf sheath of IR64and
Taiping.In TN1,gene expression was significantly low in all plant parts (Fig.5A).Transcript levels of OsLCB2a1gene were also compared at seedling,vegetative and reproductive stages of three rice varieties.The results showed that significantly higher gene expression was observed at the maximum tillering stage of IR64and Taiping (Fig.5B).
3Discussion
SPT has been suggested to be the key enzyme for the regulation of sphingolipid levels in cells.It regulates not only plant cell death but also the defense responses against non-host pathogen [24].It has been shown that LCB2a gene from serine palmitoyltransferase is required for PCD process that operates as one of the more effective strategies used as defense against pathogens in plants [25].Recent studies have noted the diversity of sphingolipid functions during plant development
and
Time after infestation/h
R e l a t i v e e x p r e s s i o n o f O s L C B 2α1 g e n e
Values are showed as mean ±standard error (n =5).Different lowercase letters indicate significant difference at the 0.05probability level.
Fig.3Analysis of OsLCB2a1expression under BPH infestation and wounding
inducement
A
B
1614
12R e l a t i v e e x p r e s s i o n l e v e l & r e s i s t a n c e s c o r e
R e l a t i v e e x p r e s s i o n l e v e l
Rice varieties
T N 1
H y b
r i d
S y 6
3W u y u g e n 2X i u s h u i 11G u a n g l u ’a i I R 64T a
i p i n g M u d g o 1086420
1
23Resistance score
456789
A:Expression of OsLCB2a1gene and resistance score of eight different rice varieties;B:Correlation between gene expression level and BPH infestation reaction score.Values are showed as mean ±standard error (n =5).Different lowercase letters indicate significant difference among the varieties infested by BPH at the 0.05probability level.The resistant varieties scored 1to 5,and the susceptible varieties scored 7to 9.
Fig.4OsLCB2a1gene expression level and BPH resistance score and their correlation in eight different rice varieties
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stress responses.In plants,LCBs act as bioactive molecules in the immune response [15].In Arabidopsis ,fuminosin B1-induced PCD is activated by increased levels of LCB,as demonstrated by a mutation in the LCB1gene that encodes one of the two subunits of SPT [26].In this study,we characterized the rice SPT genes and studied the gene expression of one SPT gene,OsLCB2a1.Multiple alignment showed that OsLCB2a1has a conservative GTFTKSFG motif corresponding to the known PLP-binding site like N.banthamiana ,A.thaliana ,S.cerevisiae,Homo sapiens and other species [17,27,19].The GTFTKSFG motif is completely conservative among the predicted LCB2proteins from different organisms,suggesting that these LCB2
polypeptides constitute catalytic subunits of SPT.For example,the pyridoxal 5′-phosphate binding site of N.banthamiana LCB2is required for its function as a cell death inducer [24].TAKAHASHI and his colleagues hypothesized that over-expression of NbLCB2enhanced the SPT activity,which in turn triggered plant cell death [24].Bioinformatics analysis showed that the SPT gene has high homology with a large number of species from bacteria to humans.
Investigations on the gene expression in different varieties,plant parts and growth stages of rice with BPH infestation will help us predict the functions of SPT gene in herbivore defense.We found that OsLCB2a1expression was up-regulated when plants were infested by BPH,suggesting that OsLCB2a1had effects on herbivore defense.Gene expression was higher in resistant varieties than in susceptible ones.The transcript
level of OsLCB2a1gene was also found to be higher in the leaf blade and leaf sheath of resistant varieties than
the root.This is probably because BPH sucks sap from leaf blade and leaf sheath of rice plants and is not a root feeder.The mRNA level of OsLCB2a1was found to be higher in maximum tillering stage of resistant varieties,which suggests that this gene is potentially more effective in the vegetative stage.
4Conclusions
The rice SPT genes were cloned in this study,which has a high degree of sequence and structure similarity with other plant SPT genes.Our data suggest that OsLCB2a1is involved in herbivore defense.Further studies are warranted to explore the function of SPT as a defense element in the interactions between rice and other pests,and to determine the underlying mechanisms of SPT effecting on pest performance.
Acknowledgements
We thank Professor K.L.Heong for his
critical modification of the earlier version of the manuscript.References:
[1]SPERLING P,HEINZ E.Plant sphingolipids:Structural
diversity,biosynthesis,first genes and functions.
Biochimica
A 0.0
IR64Taiping Rice varieties
Different stages of rice plant
TN1
Seedling
Early tillering Maximum tillering
Flowering Booting
0.51.01.52.02.53.03.54.0
4.5R e l a t i v e e x p r e s s i o n l e v e l
R e l a t i v e e x p r e s s i o n l e v e l
B 0
12345678
9Values are showed as mean ±standard error (n =5).Different lowercase letters indicate significant difference among three varieties infested by BPH at the 0.05probability level.
Fig.5OsLCB2a1gene expression level at different plant parts (A)and different growth stages (B)of rice
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371
et Biophysica Acta(BBA):Molecular and Cell Biology of Lipids,2003,1632(1/2/3):1-15.
[2]LYNCH D V,DUNN T M.An introduction to plant
sphingolipids and a review of recent advances in understanding their metabolism and function.New Phytologist,2004,161(3):677-702.
[3]MERRILL JR A H,WANG M D,PARK M,et al.(Glyco)
sphingolipidology:An amazing challenge and opportunity for systems biology.Trends in Biochemical Sciences,2007, 32(10):457-468.
[4]MERRILL A H,STOKES T H,MOMIN A,et al.
Sphingolipidomics:A valuable tool for understanding the roles of sphingolipids in biology and disease.Journal of Lipid Research,2009,50(Suppl.):97-102.
[5]PRUETT S T,BUSHNEV A K,HAGEDORN M,et al.
Thematic review series:Sphingolipids.Biodiversity of sphingoid bases(“sphingosine”)and related amino alcohols.Journal of Lipid Research,2008,49(8):1621-1639.
[6]HANNUN Y A,OBEID L M.Principles of bioactive lipid
signalling:Lessons from sphingolipids.Nature Reviews Molecular Cell Biology,2008,9(2):139-150.
[7]PAYNE S G,MILSTIEN S,SPIEGEL S.Sphingosine-1-
phosphate:Dual messenger functions.FEBS Letters,2002, 531(1):54-57.
[8]CHALFANT C E,SPIEGEL S.Sphingosine1-phosphate and
ceramide1-phosphate:Expanding roles in cell signaling.
Journal of Cell Science,2005,118(20):4605-4612.
[9]LYNCH D V,FAIRFIELD S R.Sphingolipid long-chain
base synthesis in plants(characterization of serine palmitoyltransferase activity in squash fruit microsomes).
Plant Physiology,1993,103(4):1421-1429.
[10]ZAUNER S,TERNES P,WARNECKE D.Biosynthesis of
sphingolipids in plants(and some of their functions)// Sphingolipids as Signaling and Regulatory Molecules.New York,USA:Springer,2010:249-263.
[11]DUTILLEUL C,BENHASSAINE-KESRI G,DEMANDRE
C,et al.Phytosphingosine:Phosphate is a signal for AtMPK6activation and Arabidopsis response to chilling.
New Phytologist,2012,194(1):181-191.
[12]LI S F,SONG L Y,YIN W B,et al.Isolation and functional
characterization of the genes encodingΔ8-sphingolipid desaturase from Brassica rapa.Journal of Genetics and Genomics,2012,39(1):47-59.
[13]TANG J,MENG X J,LIU H,et al.Antimicrobial activity of
sphingolipids isolated from the stems of cucumber (Cucumis sativus L.).Molecules,2010,15(12):9288-9297. [14]WU J X,LI J,LIU Z,et al.The Arabidopsis ceramidase
AtACER functions in disease resistance and salt tolerance.
The Plant Journal,2015,81(5):767-780.
[15]RIV AS-SAN VICENTE M,LARIOS-ZARATE G,PLASEN-
CIA J.Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and
compromises resistance to Alternaria alternata f.sp.
lycopersici.Planta,2013,237(1):121-136.
[16]BERKEY R,BENDIGERI D,XIAO S.Sphingolipids and
plant defense/disease:The“death”connection and beyond.
Frontiers in Plant Science,2012,3:68.
[17]BEGUM M A,SHI X X,TAN Y,et al.Molecular
characterization of rice OsLCB2a1gene and functional analysis of its role in insect resistance.Frontiers in Plant Science,2016,7:1789.
[18]YOSHIDA S,FORNO D A,COCK J boratory
Manual for Physiological Studies of Rice.Los Banos, Philippines:International Rice Research Institute,1971:61.
[19]TAMURA K,PETERSON D,PETERSON N,et al.
MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,2011, 28(10):2731-2739.
[20]TANG Q Y,ZHANG C X.Data Processing System(DPS)
software with experimental design,statistical analysis and data mining developed for use in entomological research.
Insect Science,2013,20(2):254-260.
[21]祝增荣,唐启义,吴慧明,等.现代植物保护信息技术实验.
杭州:浙江大学出版社,2015:135.
ZHU Z R,TANG Q Y,WU H M,et al.Experiments in Modern Information Techniques in Plant Protection.
Hangzhou:Zhejiang University Press,2015:135.(in Chinese)
[22]LIV AK K J,SCHMITTGEN T D.Analysis of relative gene
expression data using real-time quantitative PCR and the 2－ΔΔC T method.Methods,2001,25(4):402-408.
[23]HUANG Z,HE G,SHU L,et al.Identification and
mapping of two brown planthopper resistance genes in rice.Theoretical and Applied Genetics,2001,102(6/7):929-934.
[24]TAKAHASHI Y,BERBERICH T,KANZAKI H.Serine
palmitoyltransferase,the first step enzyme in sphingolipid biosynthesis,is involved in nonhost resistance.Molecular Plant-Microbe Interactions,2009,22(1):31-38.
[25]SAUCEDO-GARCÍA M,GUEV ARA-GARCÍA A,GONZ-
ÁLEZ-SOLÍS A,et al.MPK6,sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis.New Phytologist,2011,191(4): 943-957.
[26]SHI L H,BIELAWSKI J,MU J Y,et al.Involvement of
sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis.Cell Research,2007,17(12):1030-1040.
[27]HANADA K.Serine palmitoyltransferase,a key enzyme of
sphingolipid metabolism.Biochimica et Biophysica Acta (BBA):Molecular and Cell Biology of Lipids,2003,1632(1/ 2/3):16-30.
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