2013 Vol. 26, No. S1
The selection of suitable reference gene is an important prerequisite for successful gene expression analysis by quantitative real-time PCR (qPCR). The authors investigated the expression stability of 12 endogenous house-keeping genes (ACT4, APm, Chc, Gapc, RPL1, RPL2, EF1, EF2, eIF, E3UL, UBQ and UPL2) in 31 samples of Larix kaempferi (Lamb.) Carr. including needles, stems, roots, pollens, zygotic embryos and somatic embryos. The GeNorm and NormFinder algorithms analysis reveals that the APm is the most stable gene among different organs and developmental stages (somatic embryogenesis and seed germination), EF1 and eIF are most stable among stems and needles during plant growth (90 days, 1.5 years, 5 years, 10 years, 25 years and 50 year), respectively. The results would provide optimum internal reference genes for larch gene expression analysis using qPCR.
The seed of Larix kaempferi (Siebold. & Zucc.) Gordon was used as material to investigate the potential molecular regulation mechanisms of miRNA during seed germination. The germination process of L.kaempferi seed includes the following: The imbibition of dry seed was completed after 24 hours of socking; the testa rupture occurred after 5 days and followed by radicle emergence (videlicet, endosperm rupture) after 9 days of germination culture; the cotyledons expanded fully after 28 days. The expression level of 18 miRNAs was relatively quantified using qRT-PCR technology among five key seed germinating stages, which including dry seed (0 day after germinating), imbibed seed (1st day after germinating), testa rupture (5th day), radicle emergence (9th day) and cotyledon extension (28th day). The results are as follows. (1)The expression levels of 18 miRNAs were the highest at dry seed stage and then went down rapidly at imbibed seed and testa rupture stage. The miRNAs were highly expressed in dry seed stage than in subsequent stages. The expression level in dry seed stage was 4.8 to 43.5 times, 17.2 to 1 000 times, 45.5 to 1 000 times and 62.5 to 1 862.2 times that of those in imbibed seed, testa rupture, radicle emergence and extension cotyledon stage, respectively. (2)The expression level of miR894 was the highest and that of miR408 was the lowest in each stage; and the others fluctuated, ranking from high to low as miR951, miR950, miR156, miR159, miR396, miR398, miR390, miR160, miR947, miR165, miR319, miR162, miR171, miR397, miR395 and miR172. (3)The target genes and their possible functions predicted using bioinformatics methods were responsible for the expression of a great number of binding proteins, cellular components and MAP kinase proteins associated with seed germination. (4) JR160237 was the target gene of miR160, and the cleaved fragments mapped exactly to the predicted cleavage site (between nucleotides 10 and 11 from the 5'end of miR160). JR160237 mRNA reached the peak at imbibed seed stage, then decreased and dynamically expressed at following stages with small fluctuation.
Based on transcriptome sequencing information, the 4CL gene containing 1 537 bp coding sequences was identified from 11 types of Larix kaempferi cross combinations. Sequence analysis indicated that the coding sequence contained a complete 1 368 bp-ORF fragment, and encoded 455 amino acids. Analysis of qRT-PCR showed that the expression of 4CL in Larix82×Larix13, Larix40×Larix10 and Larix82×Larix61 offsprings were higher than that in their corresponding parent lines, except Larix16×Larix61. The results suggests that the differential expression of 4CL genes in different L. kaempferi cross combinations may be involved in the lignin biosynthesis. In addition, 66 SNPs were identified in 4CL gene, among which 45 SNPs belong to transition type, accounting for 68.18% of the total variance; 20 SNPs belong to transversion type, accounting for 30.30% of the total variance and transition: transversion = 9:4. In the transition type, A/G and C/T transition accounted for 28.79% and 39.39%, respectively; in the types of transversion, A/C, A/T, G/C, and G/T account for 4.55%, 9.09%, 9.09% and 7.58%, respectively. Moreover, the phylogenetic analysis of 4 CL gene in all materials was conducted by NJ method. All results indicated that the 4CL gene showed abundant genetic diversity.
Glutathione reductases (GRs) are key enzymes in plant glutathione-ascorbate cycle, and play important roles in plant oxidative stress tolerance. In this study, a GR gene (LaGR) was cloned from Larix kaempferi. This gene encodes protein of 563 amino acid residues, with calculated molecular masses of 61.06 kDa. RT-PCR reveals that LaGR is a constitutive expression gene, which is expressed in all tissues detected, including bud, mature needle, phloem of stem and root. The chloroplast localization of LaGR was confirmed by transient expression in Arabidopsis protoplast. LaGR was overexpressed in Escherichia coli, and purified by Ni-affinity chromatography. The purified LaGR proteins showed high catalytic activities and affinities towards substrates GSSG and NADPH. LaGR protein is a thermostable protein, and has optimal pH ranging from 7.0 to 9.0. Heavy metal ions (Cd2+, Pb2+ and Cu2+) could inhibit the LaGR's activities. When the His528 of LaGR protein was replaced by Gln, the mutant proteins showed decreased enzymatic activities and increased affinity towards substrates GSSG and NADPH. Moreover, the mutant protein showed less thermostability than wild-type protein. Thus, His528 of LaGR protein might contribute to the enzyme's catalytic activity and structural stability.
Using total RNA extracted by CTAB method as templates, the expression profiles of 5 miRNAs in young and mature leaf organs of Populus×euramericana cl.‘Zhonglin46’were studied by real-time stem-loop RT-PCR. The results showed that the expression of miR159, miR167, miR171, and miR319 in triploid young leaves were higher than those in diploid, however, the expression of miR164 in mature leaves of triploid was higher than that in diploid. In addition, the expression analysis of the corresponding miRNA target genes showed that the expression of the miRNAs and their corresponding targets were negative correlation in diploid and triploid P.×euramericana cl.‘Zhonglin46’. these findings suggested that these miRNA target genes may be related to the formation of fast-growing traits showed by triploid Populus.
According to the preliminary results of microarray analysis, a high expression gene, PtSIP3, was cloned from Populus euramericana triploid, which generated from crosses between the maternal parent P. deltoides and the pollen parent P. nigra. The binary expression vector, pRI101- PtSIP3, was constructed and transformed into Arabidopsis. So far, the PtSIP3 transgenic plants were observed the remarkable phenotypic changes with inhibition of embryo and capsule development, improvement root development. Therefore, It is speculated that the rises of PtSIP3 expression may participate in the regulation of vegetative growth and promote root development in P. euramericana triploid.
The expression patterns of 5 miRNAs(cin-miR157a, cin-miR159a, cin-miR165a, cin-miR172b and cin-miR396a) and their targets during different organs in different developmental stages were assessed by qRT-PCR. The results showed that the expression patterns of all the miRNAs are of temporal and spatial specificity. 4 miRNAs(cin-miR159a, cin-miR165a, cin-miR172b and cin-miR396a) are highly expressed in vegetative organs of mature period; one miRNA (cin-miR157a) is highly expressed in juvenile stage and reproduction organs of mature period. The results also showed that three miRNAs (cin-miR157a, cin-miR172b and cin-miR396a) and their target genes were related to the growth and decline in different developmental stages and different organs. High expression of miRNA and its target gene expression are often low. When the miRNA expression is low, its target gene expression is often high. This result indicates that miRNA may play an important role during the growth and development of C. intermedia.
The ESTs sequences of caffeic acid O-methyltransferase from Larix kaempferi transcriptome database, an cDNA clone encoding COMT, was isolated from L. kaempferi by gene-specific PCR amplification. The COMT clone was 1 350 bp in length with an open reading frame (ORF, 1 092 bp) which would be capable to encode a protein of 364 AA. The sequence indicated that the deduced amino acid contained five highly conserved regions of COMT family, and shared 94% identity with PpCOMT from Pinus pinaster. The genomic sequences of LkCOMT in 40 individuals were sequenced, and single nucleotide polymorphisms (SNPs) diversity of LkCOMT was analyzed using the software DnaSP5.0. A total of 92 SNPs were detected, and the frequency and diversity of SNPs (πT) were 1/17 bp and 0.007 80, respectively. There were 65 transition and 27 transversion of mutation types. A total of 58 SNPs were detected in the coding regions of LkCOMT, of which 15 were synonymous mutation and 37 were missense mutation.
Based on RNA sequences information of Larix kaempferi, 1 788 EST-SSR loci were found by SSRIT, with an average length of 17.5 bp. The most abundant SSRs within Larix were hexanucleotide and trinucleotide, followed by pentanucleotide, dinucleotide, and tetranucleotide. At the meantime, 656 kinds of repeat primitives were detected. In this study, a total of 500 primers were designed by "Primer Premier 5.0" and 102 pairs were synthesized. These SSR primers were used to assess the amplification efficiency of Taxaceae, Cupressaceae, and Pinaceae. The results showed that 95% and 50% of all primers could be amplified successfully in Taxaceae and Cupressaceae. And in Pinaceae, the transferability of Larix SSR in Larix, Pseudotsuga, Abies, Cedrus, Picea and Pinus was 99.01%, 67.4%., 67.4%, 63.8%, 70.2%, and 75.7% respectively. Seven different varieties of Larix genetic relationship were analyzed by using 101 pairs of SSR primers. 79 polymorphism loci were obtained, and Larix was divided into four groups at the genetic similarity coefficient of 0.69.
The methylation-sensitive amplification polymorphism (MSAP) was used to analyze the methylation in triploid and diploid poplar trees. 745 loci were detected by gel electrophoresis. The total methylation ratios in different ploidy of poplar trees varied from 13.69% to 18.84%. And the average total methylation ratios were 14.71% for diploid and 17.72% for triploid trees. The total methylation rate of poplar populations increased with the ploidy, the semi-methylation in black poplar group also increased with the ploidy, while the opposite result was found in white poplar group. No regular pattern was found in the variation of total methylation ratios of black poplar group, and the total methylation rate of white poplar group increased with the ploidy. There exists a correlation between total methylation ratios and polyploidy level in poplar ploidy group (r=0.835, Pr=0.651, P<0.05). But no significant correlation between hemimethylation ratios and polyploidy level was observed.
Based on the data of Japanese larch Solexa transcriptome sequencing obtained by splicing uridine diphosphate glucose dehydrogenase (UDPGDH) of the original sequence, the sequence ends with primers designed in parental and offspring young larch needles leaves, the cDNA sequence of UDPGDH gene was successfully cloned. Sequence analysis showed that the gene contained an open reading frame (GenBank No.KF499528) of 1 443 bp encoding a putative protein o f 480 amino acids. The expression of parental and offspring UDPGDH gene was analyzed by qRT-PCR. Offspring has a much higher expression compare with their parents' and orthogonal offspring UDPGDH gene express higher than the reciprocal cross progeny. UDP-glucose dehydrogenase is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. It might support that UDPGDH gene's high expression is beneficial to the heterosis of offspring.
Taking the shoot and the dormant bud from fine clone plants of hybrid larch(Larix kaempferi Riyong 8×L.olgensis 18-10), which is possessed of good variety for both growth and rooting, the tissue culture experiment of shoot growth and bud propagation was conducted. The effect of 15% sodium hypochlorite for 10 mins on sterilization treatment results the best, with a high rate of aseptic survival up to 91.7%. Medium B is suitable for elongation growth of the explant. While the medium BEMB is suitable for axillary buds induction and two axillary buds sprout on one shoot. Among the treatment of induction axillary buds to become branches, the BEMB, of which the content rate of nitrate nitrogen/ammonium nitrogen is 6: 1, without exogenous chemical growth regulators, has the best induction effects and the rate of branches formation is 36.5%. Dormant buds for adventitious buds induction was planted on MCM medium with different cytokinin treatment, and the results showed MCM + 1.5 mg·L-1 ZT + 0.15 mg·L-1 KT is the best medium for adventitious buds induction. The rate of buds induction is 39.1% and the average buds number induced by a bud is 2.6.
Fragments of eight specific DNA methyltransferases belonging to MET, CMT, DRM and DNMT2 were isolated from triploid Populus euramericana by using homology cloning techniques. The homology analysis indicated that five out of eight DNA methyltransferases showed 100% homology with the corresponding sequences of diploid, and splicing changes in other three genes were detected. Real-time quantitative PCR analysis detected the difference in transcript expression of these eight DNA methyltransferases in different materials, different tissues and different growth phases. Further analysis indicated that the DNA methylation status in the same tissues of different ploidy poplars was involved in different expression of different DNA methylation. Based on the results of analyzing the transcript expression of these DNA methyltransferases in different growth phases, it suggested that the members of MET gene family (PnD1 and PnD2) and CMT gene family (PnD3 and PnD4) may participate in antagonistic regulation and coordinate regulation, respectively. In addition, the results indicated that the PnD6 which belongs to DNMT2 gene family was weakly expressed in different tissues except in the stem tip of triploid P. euramericana. It implied that PnD6 may play an important role in the fast-growing of triploid P. euramericana. All these results suggested that DNA methyltransferases gene may be involved in the entire process of leaf morphogenesis.
During the process of evolution and formation of new plant species, the hybridization of two parent lines can produce F1 offspring with more excellent traits e.g. fast growing, high yielding and strong environmental adaptability. This is named heterosis or hybrid vigor. In addition, based on the polyploidization of homologous or heterogenous genomes, the polyploid plants can aslo show some traits which are superior to their parent lines. Consequently, hybridization and polyploidization have been regarded as the most important driving forces to promote plant evolution and formation of species. Moreover, heterosis and polyploidization have been widely used in agriculture, and bring enormous economic and social value. It suggests that it is very important and valuable to explore the nature basis of heterosis and polyploidization in plants which have attracted much attention. During the past years, rapid progress was made in the research associated with heterosis and polyploidization. This review aims at summarizing the research progress, and discussing the present and future research on heterosis and polyploidization of woody plants.
Non-coding small RNAs (ncsRNAs) are endogenous RNA molecule, 20—26 nucleotides in length, which have been found in diverse plants and function as transcriptional and post-transcriptional regulators of gene expression. The microRNAs (miRNAs) and other ncsRNAs play essential roles in development and physiologic process of plants. The miRNAs and other ncsRNAs are conserved among terrestrial plants, and the innovation of miRNAs and other ncsRNAs appears to be associated with the advent of evolution lineages of terrestrial plants, indicating that ncsRNAs have huge impacts on plant phylogeny. This review summarizes the conservation of miRNAs and other ncsRNAs, and their functions in plants. The vital roles of ncsRNAs during the in the evolution of terrestrial plants are also discussed.
Floral development is a complicated morphogenesis process. A good comprehension towards the floral development mechanism of forest trees is important for genetic manipulation of forest trees. This review summarizes the research advance on the molecular mechanism of trees' floral induction and cases of artificially regulating trees' reproductive development through transgenetic methods and other approaches. The outlook of those approaches which interfere the floral induction and reproductive development in forest tree breeding is also discussed.
Populus euramericana‘MR3’is a transgenic clone transformed with five foreign genes (JERF36, SacB, vgb, and BtCry3A+ OC-1) via genetic engineering approach using Populus ×euramericana‘Guariento’as the receptor. This variety inherits the features of fast-growing and broad adaptation that the receptor possessed and further enhances the tolerance to various stresses including drought, salt, flooding, and insects feeding. The experiments in greenhouse for stress tolerance assays indicated that the expression of foreign genes led to improved tolerance to drought, salt, flooding, and insects (mainly Plagiodera versicolora). Field trial in an area with salinized soil in Shandong Province showed a 7.77% increase in stem volume than that of the control clones of 5-year-old saplings. Another field trial at Linghai in Liaoning Province indicated a 15.41% increase in stem volume than that of the control clones of 2-year-old. In the experiments in greenhouse, laboratory, and field, the P. euramericana‘MR3’showed enhanced characteristics such as fast-growing, higher tolerance to drought, salt, insects, and flooding stress, and is expected to be one of the ideal new poplar varieties for wood industry and ecosystem conservation in North China.