| The full-length of four cDNA of Lhca genes, LhcaPe01 (GenBankEU035496), LhcaPe02 (GenBank EU121593), LhcaPe03 (GenBank EU366147), LhcaPe04 (GenBank EU366146), were cloned from the first strand of Moso bamboo cDNA through RT-PCR and RACE methods. Its nucleotide sequences and the encoded amino acid sequences were analyzed and the protein structure was predicted via bioinformatics approach. To know the function of four genes of bamboo PSI, different light intensity of the Moso bamboo were dealt with based on the change in fluorescence parameters to determine the time for collecting leaves, and fluorescence quantitative real-time PCR was carried out by choosing of GAPDH andβ-actin genes as housekeeping genes and natural leaves as contrast, and the relative expressive quantity of Lhca genes were calculated through 2-ΔΔCt method. In addition, the four relative expressive quantities of Lhca genes in the same period of the different tissues were also calculated. Through SYBR-GreenΙand melting curve this dissertation detects of gene expression of members of the Lhca genes family. The following conclusions were drawn:(1)The length of LhcaPe01 is 1051 bp, which contains an open reading frame encoding 246 amino acids ranging from 39 bp to 779 bp. There was a 38 bp untranslated region at the 5′end, and a 242 bp untranslated region and 30 bp Poly (A) at the 3′end. From the analysis of the sequence through the website of ExPASy, The DNA sequence two 4Fe-4S and two 2Fe-2S ferredoxins signal regions associated with the photosynthesis signature. The gene-encoded amino acid sequence contains three Protein kinase C phosphorylation sites, two Casein kinaseⅡphosphorylation sites, five N-myristoylation sites and two Chlorophyll a/b binding protein and a yiaA/B two helix domain. In addition, the pI and Mw of protein encoded by LhcaPe01 were predicted to be 5.4000 and 22107.34 Da respectively.(2)The length of LhcaPe01 is 1100 bp, which contains an open reading frame encoding 262 amino acids ranging from 74 bp to 862 bp. There was a 73 bp untranslated region at the 5′end, and a 234 bp untranslated region and 14 bp Poly (A) at the 3′end. The pI and Mw of protein encoded by LhcaPe02 were predicted to be 6.14 and 28095.11 Da. Such a DNA sequence associated with the photosynthesis signature has two 4Fe-4S ferredoxins signal regions. The gene-encoded amino acid sequence contains an Amidation site, three Protein kinase C phosphorylation sites, two Casein kinaseⅡphosphorylation sites, five N-myristoylation sites and a Chlorophyll a/b binding protein domain.(3)The length of LhcaPe03 is 1149 bp, which contains an open reading frame encoding 262 amino acids ranging from 76 bp to 861 bp. There was a 75 bp untranslated region at the 5′end, and a 288 bp untranslated region and 27 bp Poly (A) at the 3′end. The pI and Mw of protein encoded by LhcaPe03 were predicted to be 5.08 and 27686.79 Da respectively. The DNA sequence has two 4Fe-4S and three 2Fe-2S ferredoxins signal regions. The gene-encoded amino acid sequence contains a Protein kinase C phosphorylation site, a Casein kinaseⅡphosphorylation site, a N-myristoylation site and a Chlorophyll a/b binding protein domain.(4)The length of LhcaPe04 is 1071 bp,which contains an open reading frame encoding 245 amino acids ranging from 1bp to 738 bp. There was no untranslated region cloned at the 5′end, and a 304 bp untranslated region and 29 bp Poly (A) at the 3′end. The pI and Mw of protein encoded by LhcaPe04 were predicted to be 6.95 and 26898.83 Da respectively. The DNA sequence has two 2Fe-2S ferredoxins signal regions. The-gene encoded amino acid sequence contains seven Protein kinase C phosphorylation sites, an N-myristoylation site, an N-glycosylation site and a Chlorophyll a/b binding protein domain.(5)The barley and rice was the highest in similarity, which is drawn from the analysis through NCBI Blast to four PSI genes. The similarity of Lhca DNA sequence is higher than that of encoding amino acid sequence. The secondary structure of LhcaPe01 is significantly different from that of three other genes, and the differences among LhcaPe02, LhcaPe03 and LhcaPe04 are quite few. According to the prediction and simulation through SWISS MODEL, the protein structural models of four Lhca genes of bamboo, barley, rice and maize are similar. The differences between the four types of protein in different plant species are bigger than that of different type protein in the same plant species, in particular, the conformation of Lhca4 is different from that of three other proteins.(6)The Lhca1and Lhca2 similarity of P. iridescens, P.fimbriligula and P.edulis is very high. The DNA sequence identity distance of Lhca1 and Lhca2 is over 99% and 98% respectively in Phyllostachys. In general, the similarity of three bamboo species Lhca1 and Lhca2 genes in Phyllostachys is higher than rice and barley, which proved that Lhca1 is more conservative than Lhca2.(7)In young leaves, old leaves, culm tips, culms and bamboo shoots, Lhca1, Lhca2, Lhca3 and Lhca4 all have their expressions, which are different in different tissues. Lhca1 gene in young leaves of the expression is the highest; Lhca2 in old leaves is the highest; Lhca3 and Lhca4 in culm tips is the highest. The expression of Lhca1 in young leaves is the highest and Lhca3 is lowest in the bamboo shoots in all the samples. Four genes expression in bamboo shoot is quite high.(8)The expression orders of four genes of the leaves under light treatment are all Lhca1> Lhca4> Lhca2> lhca3. The expression is down regulated in strong light, and up regulated in low light. In light intensity of 3000μmol·m-2·s-1, the mRNA levels of Lhca1-4 is 89.5%, 30.6%, 48.3%, 63.9% of the normal level respectively; the order of reduction is Lhca2> Lhca3> Lhca4> Lhca1. In light intensity of the 2000μmol·m-2·s-1, the mRNA levels of Lhca1-4 is 27.9%, 17.8%, 69.7% and 35.1% of the normal level respectively, the order of reduction is Lhca2> Lhca1> Lhca4> Lhca3. In light intensity of the 1600μmol·m-2·s-1, the mRNA levels of Lhca1-4 is 73.3%, 51.8%, 35.7%, 85.4% of the normal level respectively, the order of reduction is Lhca3> Lhca2> Lhca1> Lhca4.The relative expression levels increase through the treatment of low light and medium light after 20d. Four Lhca genes expression is up regulated in light intensity of 1000μmol·m-2·s-1 and their values are 116.7%, 115.4%, 187.9%, 102.4% compared with that in the nature light, the order of increase is Lhca1> Lhca4> Lhca3> Lhca2. The expression of Lhca1, Lhca3, Lhca4 genes is up regulated and Lhca2 gene expression is invariable in light intensity of 500μmol·m-2·s-1, and their values are 237.4%, 99.1%, 122.2%, 147.5% compared with that in the nature light, the order of increase is Lhca1> Lhca4> Lhca3> Lhca2. Four Lhca genes expression is up regulated in light intensity of 10μmol·m-2·s-1 and their values are 607.2%, 168.5%, 219.2%, 149.6% compared with that in the nature light, the order of increase is Lhca1> Lhca3> Lhca2> Lhca4. Four Lhca genes expression is up regulated in light intensity of 10μmol·m-2·s-1 and their values are 607.2%, 168.5%, 219.2%, 149.6% compared with that in the nature light, the order of increase is Lhca1> Lhca3> Lhca2> Lhca4.Four genes mRNA leves drop all along from 1 d to 15 d in light intensity of 1600μmol·m-2·s-1. Lhca mRNA levels rapid increase through the treatment of low light and medium light from 1d to 6 d but the enhancement is repressed to 20 d. |
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